Ehrlichia canis DIVA (differentiate infected from vaccinated animals)

ABSTRACT

The invention provides  Ehrlichia canis  antigens that can be used to detect  E. canis  infected animals regardless of whether the animals have been vaccinated for  E. canis . The invention also provides compositions and methods for determining the presence of  E. canis  antigens and antibodies.

PRIORITY

This application is a divisional application of U.S. Ser. No. 12/262,709(now U.S. Pat. No. 7,888,054), which was filed on Oct. 31, 2008, whichclaims the benefit of U.S. Ser. No. 60/984,019, which was filed on Oct.31, 2007. These applications are incorporated herein by reference intheir entirety.

SEQUENCE LISTING

This document incorporates by reference an electronic sequence listingtext file, which was electronically submitted along with this document.The text file is named “04947DUSDIVs12011.txt” is 84.8 kb, and wascreated on Apr. 4, 2011.

BACKGROUND OF THE INVENTION

The Ehrlichia are obligate intracellular pathogens that infectcirculating white blood cells in mammalian hosts. Ehrlichia canis caninfect canines and humans and cause canine monocytic ehrlichiosis (CME)and human monocytic ehrlichiosis (HME), respectively. The canine diseaseis characterized by fever, lymphadenopathy, weight loss, andpancytopenia. In humans the disease is characterized by fever, headache,mylagia, and leukopenia. Early detection and treatment are important fortreating both canine and human ehrlichiosis.

SUMMARY OF THE INVENTION

In one embodiment the invention provides a method of distinguishingbetween animals that have been (a) infected with Ehrlichia canis; and(b) animals that have not been infected with E. canis regardless ofwhether the animal has been vaccinated for E. canis. The methodcomprises

-   -   (a) contacting a biological sample from an animal with one or        more first purified polypeptides that do not specifically bind        to antibodies that are a component of the animal's immune        response to an E. canis vaccine; wherein the one or more first        purified polypeptides have at least 95% identity to SEQ ID        NOs:22-33 and wherein the one or more first purified E. canis        polypeptides specifically bind an antibody that is specific        for E. canis; and    -   (b) detecting whether antibodies in the biological sample        specifically bind to the one or more first purified E. canis        polypeptides.        If antibodies in the biological sample specifically bind to the        one or more first purified polypeptides, then the animal is        infected with E. canis. The one or more first purified        polypeptides can be about 15 to about 75 amino acids in length.        The one or more first purified polypeptides can be linked to a        heterologous amino acid sequence, an indicator reagent, an amino        acid spacer, an amino acid linker, a signal sequence, a stop        transfer sequence, a transmembrane domain, a protein        purification ligand, or a combination thereof. The method can        further comprise determining whether antibodies in the        biological sample specifically bind to one or more second        purified E. canis polypeptides that are an element of an E.        canis vaccine. If antibodies in the biological sample        specifically bind to the one or more first purified E. canis        polypeptides and specifically bind to the one or more second        purified E. canis polypeptides, then the animal has been        infected with E. canis and the vaccination status for E. canis        is unknown. If antibodies in the sample do not specifically bind        to the one or more first purified E. canis polypeptides and        specifically bind to the one or more second purified E. canis        polypeptides, then the animal has not been infected with E.        canis and has been vaccinated for E. canis. If antibodies in the        sample do not specifically bind to the one or more first        purified polypeptides and do not specifically bind to the one or        more second purified polypeptides then the animal has not been        vaccinated for E. canis and has not been infected by E. canis.

Yet another embodiment of the invention provides a method of determiningan animal's vaccination and infection status for E. canis. The methodcomprises:

-   -   (a) contacting a biological sample from an animal with one or        more first purified polypeptides that do not specifically bind        to antibodies that are a component of the animal's immune        response to an E. canis vaccine, wherein the one or more first        purified polypeptides have at least 95% identity to SEQ ID        NOs:22-33 and wherein the one or more first purified        polypeptides specifically bind an antibody that is specific        for E. canis, and one or more second purified polypeptides that        specifically bind to an antibody that is a component of the        animal's immune response to an E. canis vaccine; and    -   (b) detecting whether antibodies in the biological sample        specifically bind to the one or more first purified polypeptides        and to the one or more second purified polypeptides.        If antibodies in the biological sample specifically bind to the        one or more first purified E. canis polypeptides and        specifically bind to the one or more second purified E. canis        polypeptides, then the animal has been infected with E. canis        and the vaccination status for E. canis is unknown. If        antibodies in the sample do not specifically bind to the one or        more first purified E. canis polypeptides and specifically bind        to the one or more second purified E. canis polypeptides, then        the animal has not been infected with E. canis and has been        vaccinated for E. canis. If antibodies in the sample do not        specifically bind to the one or more first purified polypeptides        and do not specifically bind to the one or more second purified        polypeptides then the animal has not been vaccinated for E.        canis and has not been infected by E. canis. The one or more        first purified polypeptides can be about 15 to about 75 amino        acids in length. The one or more first purified polypeptides can        be linked to a heterologous amino acid sequence, an indicator        reagent, an amino acid spacer, an amino acid linker, a signal        sequence, a stop transfer sequence, a transmembrane domain, a        protein purification ligand, or a combination thereof.

Still another embodiment of the invention provides a method fordetermining the presence of an antibody or antigen-binding fragmentsthereof that are specific for E. canis, in a test sample. The methodcomprises:

-   -   (a) contacting the test sample with one or more purified        polypeptides that have at least 95% identity to SEQ ID NOs:22-33        wherein the one or more purified polypeptides are about 15 to        about 75 amino acids in length, and wherein the one or more        first purified polypeptides specifically bind an antibody that        is specific for E. canis, under conditions suitable for specific        binding of the one or more purified polypeptides to the        antibodies or antigen-binding fragments thereof; and    -   (b) detecting the presence of specific binding of the one or        more purified polypeptides to the antibodies or antigen-binding        fragments thereof.        The presence of specific binding of the one or more purified        polypeptides to the antibodies or antigen-binding fragments        indicates the presence of the antibodies or antigen-binding        fragments thereof specific for E. canis in the test sample. The        one or more purified polypeptides can be linked to a        heterologous amino acid sequence, an indicator reagent, an amino        acid spacer, an amino acid linker, a signal sequence, a stop        transfer sequence, a transmembrane domain, a protein        purification ligand, or a combination thereof. The method can        further comprise detecting the amount of specific binding. The        one or more purified polypeptides can be immobilized to a solid        support.

Even another embodiment of the invention provides a compositioncomprising:

-   -   (a) one or more purified polypeptides consisting of SEQ ID        NO:22-33; or    -   (b) one or more purified polypeptides having at least 95%        identity to SEQ ID NOs:22-33 wherein the one or more purified        polypeptides are about 15 to about 75 amino acids in length, and        wherein the one or more purified polypeptides specifically bind        an antibody that is specific for E. canis;    -   (c) SEQ ID NO:33, wherein the X at position 1 is absent or C,        the X at position 4 is H or Q, the X at position 25 is D or G,        and the X at position 36 is E or G;    -   (d) amino acids 1-27 of SEQ ID NO:33, wherein the X at position        1 is C, the X at position 4 is H, the X at position 25 is D or        G;    -   (e) amino acids 13-41 of SEQ ID NO:33, wherein the X at position        25 is D or G, the X at position 36 is E or G; and a C is        optionally present at the amino terminus;    -   (f) amino acids 24-49 of SEQ ID NO:33, wherein the X at position        25 is D or G, the X at position 36 is E or G, and a C is        optionally present at the amino terminus;    -   (g) amino acids 1-27 of SEQ ID NO:33, wherein the X at position        1 is C or absent, and wherein the X at position 25 is D or G;    -   (h) amino acids 13-41 of SEQ ID NO:33, wherein the X at position        25 is D or G, the X at position 36 is E or G, and a C is        optionally present at the amino terminus;    -   (i) amino acids 24-49 of SEQ ID NO:33, wherein the X at position        25 is D or G, the X at position 36 is E or G, and a C is        optionally present at the amino terminus;    -   (j) amino acids 13-27 of SEQ ID NO:33, wherein the X at position        25 is D or G, and a C is optionally present at the amino        terminus;    -   (k) amino acids 24-41 of SEQ ID NO:33, wherein the X at position        25 is D or G, the X at position 36 is E or G, and a C is        optionally present at the amino terminus;    -   (l) amino acids 13-41 of SEQ ID NO:33, wherein the X at position        25 is D or G, the X at position 36 is E or G, and a C is        optionally present at the amino terminus;    -   (m) amino acids 24-49 of SEQ ID NO:33, wherein the X at position        25 is D or G, the X at position 36 is E or G, and a C is        optionally present at the amino terminus;    -   (n) amino acids 24-41 of SEQ ID NO:33, wherein the X at position        25 is D or G, the X at position 36 is E or G, and a C is        optionally present at the amino terminus; or    -   (o) combinations of (a)-(n).        The one or more purified polypeptides can be in a multimeric        form. The one or more purified polypeptides can be linked to a        heterologous protein, an indicator reagent, an amino acid        spacer, an amino acid linker, a signal sequence, a stop transfer        sequence, a transmembrane domain, a protein purification ligand,        or a combination thereof.

Another embodiment of the invention provides a method of generating animmune response in an animal comprising administering one or morepurified polypeptides having at least 95% identity to SEQ ID NOs:22-33or a combination thereof to the animal, wherein the one or more purifiedpolypeptides generate an immune response in the animal. The one or morepurified polypeptides can be about 15 to about 75 amino acids in length.The one or more purified polypeptides can be in a multimeric form. Theone or more purified polypeptides can be linked to a heterologousprotein, an indicator reagent, an amino acid spacer, an amino acidlinker, a signal sequence, a stop transfer sequence, a transmembranedomain, a protein purification ligand, or a combination thereof.

Still another embodiment of the invention provides a method for theprophylaxis, treatment, or amelioration of an Ehrlichia canis infectionin an animal comprising administering to the animal:

-   -   (a) one or more purified polypeptides having at least 95%        sequence identity to SEQ ID NOs:22-33, or a combination thereof;        or    -   (b) one or more nucleic acids encoding one or more purified        polypeptides comprising SEQ ID NOs:22-33, or a combination        thereof; or    -   (c) one or more antibodies that specifically bind one or more        purified polypeptides comprising SEQ ID NOs:22-33, or a        combination thereof;

whereby the E. canis infection is prevented, ameliorated, or treated.

Yet another embodiment of the invention provides a method of monitoringtreatment of an E. canis infection in a patient comprising: (a)determining the level of anti-E. canis antibodies in a first sample froma patient prior to or in the early stages of a treatment for an E. canisinfection by a method of claim 10; (b) determining the level of anti-E.canis antibodies in a second sample from the patient after treatment iseffected by a method of claim 10; and (c) comparing the amount ofanti-E. canis antibodies in the first sample with the amount of anti-E.canis antibodies in the second sample to assess a change and therebymonitor treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows SNAP® 3Dx® Assay (reversible flow chromatographic assay)evaluation of laboratory beagles. The SNAP® device used as described bymanufacturer. “Pre” sample is from day 0. “Post” sample is from day 42.The E. canis positive spot became positive in all 4 dogs for the day 42sample. Similar results were observed for the day 70 sample.

FIG. 2 shows a gel of E. canis proteins separated using 2D gelelectrophoresis. Stained with BIOSAFE™ Coomassie Blue (Bio-Rad Inc.).

FIG. 3 shows a western blot of E. canis proteins using dog seraharvested at day 0. The plasma dilution is 1:100. These dogs werenegative for reactivity with E. canis antigens.

FIG. 4 shows a western blot of E. canis proteins using dog sera from apool of four vaccinated animals. The sera dilution is 1:100.

FIG. 5 shows a western blot of E. canis proteins using dog plasma from apool of infected animals. The sera dilution is 1:1000.

FIG. 6 shows a western blot of six different E. canis DIVA antigensexpressed in E. coli and probed with either dog sera from a pool of fourinfected animals (A) or dog sera pooled from four vaccinated animals(B). Sera dilutions were 1:100 for vaccinated animals or 1:500 for theinfected animals. The DIVA antigens represented include: (1) 200 kDaantigen, (2) Ribosomal protein L1, (3a and 3b) “ATPase”—two differentsegments, (4) 120 kDa antigen, (5) Heat shock proteins/p16 antigen.

FIG. 7 demonstrates that cloned p16 antigen is recognized by sera fromdogs infected with E. canis but not those that were vaccinated (shown as“challenged sera”). Lysates from uninduced (U) or induced (I) bacteriatransformed with a vector expressing the p16 antigen or the originalgenomic fragment (+C) were separated by SDS-PAGE and transferred tonitrocellulose for western blot analysis.

FIG. 8 demonstrates detection of antibodies specific for E. canis usinga polypeptide shown in SEQ ID NO:23 in dogs over a time course includinginfection, treatment, and recovery.

FIGS. 9A-B demonstrate detection of antibodies specific for E. canisusing a polypeptide shown in SEQ ID NO:10 in two dogs that have not beenvaccinated for E. canis over a time course of infection.

FIGS. 10A-C demonstrate detection of antibodies specific for E. canisusing a polypeptide shown in SEQ ID NO:10 in three dogs that have beenvaccinated (RIBI adjuvant) for E. canis over a time course of infection.

FIGS. 11A-C demonstrate detection of antibodies specific for E. canisusing a polypeptide shown in SEQ ID NO:10 in three dogs that have beenvaccinated (RIBI+BCG adjuvant) for E. canis over a time course ofinfection.

DETAILED DESCRIPTION OF THE INVENTION

Ehrlichia canis antigens are disclosed that can be used to differentiateE. canis naturally-infected animals from animals that have beenvaccinated against with E. canis. “Vaccinated” means the administrationof a vaccine composition that can prevent or ameliorate the effects ofinfection by a pathogen by establishing or improving immunity to thepathogen. Vaccine compositions can comprise dead, inactivated orattenuated pathogens or purified products or portions of the pathogen.Vaccination is not necessarily 100% effective.

Before describing the present invention in detail, a number of termswill be defined. As used herein, the singular forms “a,” “an”, and “the”include plural referents unless the context clearly dictates otherwise.

As used herein, the term “polypeptide” refers to a compound of a singlechain or a complex of two or more chains of amino acid residues linkedby peptide bonds. The chain(s) may be of any length and can comprise afusion protein. Although “protein” is often used in reference torelatively large polypeptides, and “peptide” is often used in referenceto small polypeptides, usage of these terms in the art overlaps andvaries. The term “polypeptide” as used herein thus refersinterchangeably to peptides, polypeptides, proteins, or fusion proteinsunless otherwise noted. The term “amino acid” refers to a monomeric unitof a peptide, polypeptide or protein. The term “polypeptides” can referto one or more of one type of polypeptide (a set of polypeptides).“Polypeptides” can also refer to mixtures of two or more different typesof polypeptides (a mixture of polypeptides). The terms “polypeptides” or“polypeptide” can each also mean “one or more polypeptides.”

Polypeptides of the invention can be “isolated.” An isolated polypeptideis a polypeptide that is not immediately contiguous with one or both ofthe amino and carboxy flanking amino acid sequences that it is naturallyassociated with. In particular, “an isolated polypeptide shown in SEQ IDNOs:22-33” means that the polypeptide is not immediately contiguous withone or both of the amino and carboxy flanking amino acid sequences thatit is naturally associated with (where the polypeptide is a naturallyoccurring polypeptide) in an E. canis protein molecule.

As used herein, “antigen” as used herein refers to a molecule againstwhich a subject can initiate a humoral and/or cellular immune response.Antigens can be any type of biologic molecule including, for example,simple intermediary metabolites, sugars, lipids, and hormones as well asmacromolecules such as complex carbohydrates, phospholipids, nucleicacids and proteins. In the compositions and methods of the invention, anantigen can be a polypeptide, e.g., one comprising at least about six ormore amino acids.

As used herein, a “derivative” of an E. canis antigen polypeptide, or anantigen or polypeptide that is “derived from” an E. canis antigen orpolypeptide, refers to a antigen or polypeptide in which the native formhas been purified, modified or altered. Such modifications include, butare not limited to: amino acid substitutions, modifications, additionsor deletions; alterations in the pattern of lipidation, glycosylation orphosphorylation; reactions of free amino, carboxyl, or hydroxyl sidegroups of the amino acid residues present in the polypeptide with otherorganic and non-organic molecules; and other modifications, any of whichmay result in changes in primary, secondary or tertiary structure.

A “biological sample” is any sample from an animal that is expected tocontain immunoglobulins. For example, a biological sample can be plasma,blood, serum, saliva, urine, feces, wound exudate, cerebrospinal fluid(CSF), semen, sputum, as well as tissue extracts, and cell extracts. Inone embodiment of the invention a test sample can be obtained from ananimal such as a horse, dog, cow, llama, sheep, goat, deer, elk, rodentor any other animal. A human is considered to be an animal herein. Theseexamples are not to be construed as limiting the sample types or animalsthat are applicable to the present invention.

An “infection,” such as in an E. canis infection, means that an animalhas been exposed to E. canis, regardless of whether the animal exhibitsclinical symptoms of E. canis. A natural infection refers to an exposurethat occurs as a result of one of the natural transmission methods forE. canis, such as transmission by ticks. An infection does not includean exposure to E. canis through vaccination.

A “polypeptide or antigen that is not an element of an E. canis vaccine”is any E. canis polypeptide or antigen that is not present in aparticular E. canis vaccine or vaccines. A “polypeptide or antigen thatis not an element of an E. canis vaccine” is also any E. canispolypeptide or antigen that is not an immunogenically active portion ofan E. canis vaccine. That is, the polypeptide or antigen may be presentin the vaccine, but an immune response against the polypeptide orantigen (e.g., the generation of antibodies that specifically bind tothe polypeptide or antigen) is not generated in response toadministration of the E. canis vaccine. Elements of the vaccine(s) canbe portions of a subunit vaccine that includes less than the entirebacterium; these portions can be chemically synthesized or expressedrecombinantly before becoming part of the vaccine, and these portionscan be encoded by one or more vectors that express an immunogeniccomposition in vivo.

An “antibody that is a component of an animal's immune response to an E.canis vaccine” refers to an antibody that is elicited as the result of avaccination with an E. canis vaccine. These antibodies can be identicalto or similar to antibodies elicited as the result of a natural E. canisinfection. These antibodies will be maintained at a sufficient titer andso as to provide a protective and neutralizing effect against thebacteria. A successful vaccination produces a measurable level of theantibody (or antibodies) that is elicited by a component of the E. canisvaccine. Examples of E. canis antigens that elicit antibodies that canbe a component of an animal's immune response to an E. canis vaccine arep28-1, p28-2, p28-3, p28-4, p28-5, p28-6, p28-'7, p28-8, p28-9 (see,U.S. Pat. Nos. 6,660,269; 6,458,942; 6,403,780; 6,392,023), proA, ProB,mmpA, cytochrome oxidase (see, U.S. Pat. Publ. 20040170972), p43 (see,U.S. Pat. No. 6,355,777), which is the N-terminal portion of p153, aglycoprotein (see, U.S. Pat. Publ. 2004/0121433), p153, and p30-1,p30-2, p30-3, p30-4, p30-5, p30-6, p30-7, p30-8, p30-9, p30-10, p30-11,p30-12, p30-13, p30-14, p30-15, p30-16, p30-17, p30-18, p30-19, p30-20(Ohashi et al. 2001, Infection and Immunity 69(4): 2083-91).

An immune response is the development in an organism of a cellularand/or antibody mediated immune response to an antigen such as apolypeptide. Usually such a response includes, but is not limited to,one or more of the following: production of antibodies, B cells, helperT cells, suppressor T cells, and/or cytotoxic T cells. An immuneresponse can be detected using any of several assays known to those withskill in the art.

Polypeptides of the Invention

Biological samples from animals that have been vaccinated against E.canis have the potential for producing a positive result in a test forE. canis infection due to the presence of antibodies produced inresponse to the vaccine. In one aspect, the invention provides a methodof distinguishing between animals that have been infected with E. canisand those that have not been infected with E. canis, regardless ofwhether the animal has been vaccinated for E. canis. Methods includecontacting a biological sample from the animal with an antigen derivedfrom E. canis that does not specifically bind to an antibody that is acomponent of the animal's antibody response to a particular E. canisvaccine, but that does specifically bind to an antibody that isgenerated in response to infection by E. canis.

The development of E. canis antibodies in an animal against a vaccine isdependent upon the particular vaccine used to vaccinate the animal. Thedifference in the immune response between animals that are vaccinatedagainst E. canis and animals that are naturally or experimentallyinfected with E. canis provides a means for determining whether ananimal is naturally or experimentally infected with E. canis, regardlessof whether the animal has been vaccinated for E. canis. Therefore, usingthe methods of the invention, animals that have been infected with E.canis can be distinguished from animals that have not been infected withE. canis and/or have been vaccinated against E. canis. Antigens of theinvention, their immunodominant regions, and epitopes can be used in themethods of the invention. These compositions can be referred to as E.canis DIVA antigens (Differentiate Infected from Vaccinated Animals). AnE. canis DIVA antigen induces an immune response, e.g., the productionof specific antibodies, in an animal that is different from the immuneresponse induced in the animal by a particular E. canis vaccine.

Accordingly, detection of specific binding between an E. canis DIVAantigen and an antibody that is not a component of an animal's immuneresponse to a particular vaccine can indicate a natural or experimentalE. canis infection. The absence of such binding can indicate the absenceof E. canis infection. In addition, a second, separate antigen, such asan E. canis antigen that specifically binds an antibody that is acomponent of an animal's immune response to a particular E. canisvaccine, can be used to detect antibodies produced in response tovaccination (herein referred to as “an E. canis vaccine antigen”). An E.canis vaccine antigen not only specifically binds an antibody that is acomponent of an animal's immune response to a particular E. canisvaccine, but can also specifically bind to antibodies that are acomponent of an animal's immune response to infection by E. canis. If anantibody specific for an E. canis vaccine antigen is detected, then theanimal has been vaccinated and/or infected. The detection of neitherantibody indicates no infection and no vaccination. As such, variouscombinations of separate capture reagents can lead to a determination ofthe vaccination and/or infection status of the test subject.

In one aspect, a method of the invention includes contacting abiological sample from an animal with an antigen that is a part of theE. canis bacteria, but is not an element of a particular E. canisvaccine. In another aspect, a method of the invention includescontacting a biological sample from an animal with an antigen that ispresent in or part of E. canis bacteria and an E. canis vaccine, whereinan immune response against the antigen (e.g., the generation ofantibodies that specifically bind to the antigen) is generated inresponse to infection with the E. canis bacteria, but not in response toadministration of the E. canis vaccine. In another aspect, a biologicalsample from an animal is analyzed to detect the presence or absence ofantibodies specific for an E. canis DIVA antigen, and the presence orabsence of antibodies specific for an E. canis vaccine antigen. It isthen determined that the animal has not been infected and has not beenor vaccinated by determining the absence of such antibodies.

In one aspect of the invention, a DIVA antigen is not an element of anE. canis vaccine. In another aspect of the invention, a DIVA antigen ispart of an E. canis vaccine, but an immune response (e.g., thegeneration of an antibody that specifically binds the DIVA antigen) isnot generated in response to administration of the E. canis vaccine. Thevaccination or infection status of an animal can be determined bydetecting whether antibodies in the sample specifically bind to one ormore E. canis vaccine antigens and whether antibodies in the samplespecifically bind to one or more DIVA antigens. If antibodies in thesample specifically bind to one or more of the vaccine antigens andspecifically bind to one or more of the DIVA antigens, then the animalis infected with E. canis and the vaccination status of the animal isunknown. If antibodies in the sample specifically bind to one or more ofthe E. canis vaccine antigens and do not specifically bind to one ormore of the DIVA antigens, then the animal is vaccinated for E. canisand is not infected with E. canis. If antibodies in the sample do notspecifically bind to one or more of the E. canis vaccine antigens and donot specifically bind to one or more of the DIVA antigens, then theanimal is not infected with E. canis and is not vaccinated for E. canis.

One aspect of the invention provides a method of distinguishing betweenanimals that have been (a) infected with Ehrlichia canis; and (b)animals that have not been infected with E. canis regardless of their E.canis vaccine status. The method comprises contacting a biologicalsample from an animal with a first purified E. canis polypeptide thatdoes not substantially specifically bind to antibodies that are acomponent of the animal's immune response to an E. canis vaccine;wherein the first purified E. canis polypeptide comprises SEQ IDNOs:22-33 or combinations thereof and detecting whether antibodies inthe sample specifically bind to the first purified E. canis polypeptide.If antibodies in the sample specifically bind to the first purified E.canis polypeptide, then the animal is infected with E. canis; and ifantibodies in the sample do not substantially specifically bind to thefirst purified E. canis polypeptide, then the animal is not infectedwith E. canis.

The method can further comprise determining whether antibodies in thesample specifically bind to a second purified E. canis polypeptide thatcomprises an E. canis vaccine antigen. If antibodies in the samplespecifically bind to one or more of the E. canis vaccine antigens andspecifically bind to one or more of the DIVA antigens, then the animalis infected with E. canis and the vaccination status of the animal isunknown. If antibodies in the sample specifically bind to one or more ofthe E. canis vaccine antigens and do not specifically bind to one ormore of the DIVA antigens, then the animal is vaccinated for E. canisand is not infected with E. canis. If antibodies in the sample do notspecifically bind to one or more of the E. canis vaccine antigens and donot specifically bind to one or more of the DIVA antigens, then theanimal is not infected with E. canis and is not vaccinated for E. canis.In one embodiment of the invention antibodies in test samples do notsubstantially specifically bind to DIVA antigens and/or E. canis vaccineantigens. Substantially no specific binding is an amount of binding thatwould be considered a negative result by one of skill in the art.

One aspect of the invention provides a method of determining an animal'svaccination and infection status for E. canis. The method comprises:

-   -   (a) contacting a biological sample from an animal with a first        purified polypeptide that does not substantially specifically        bind to antibodies that are a component of the animal's immune        response to an E. canis vaccine, wherein the first purified        polypeptide comprises SEQ ID NOs:22-33 or combinations thereof,        and a second polypeptide that specifically binds to an antibody        that is a component of the animal's immune response to an E.        canis vaccine;    -   (b) detecting whether antibodies in the sample specifically bind        to the first and second purified polypeptides;        wherein if antibodies in the biological sample specifically bind        to the one or more first purified E. canis polypeptides and        specifically bind to the one or more second purified E. canis        polypeptides, then the animal has been infected with E. canis        and the vaccination status for E. canis is unknown; wherein if        antibodies in the sample do not specifically bind to the one or        more first purified E. canis polypeptides and specifically bind        to the one or more second purified E. canis polypeptides, then        the animal has not been infected with E. canis and has been        vaccinated for E. canis; and wherein if antibodies in the sample        do not specifically bind to the one or more first purified        polypeptides and do not specifically bind to the one or more        second purified polypeptides then the animal has not been        vaccinated for E. canis and has not been infected by E. canis.

Table 1 demonstrates the infection and/or vaccination status of animalsthat can be determined with E. canis DIVA antigens and E. canis vaccineantigens. “Not Done” in Table 1 means that a particular test was notcompleted and therefore no result is available. For example if abiological sample from an animal is tested with an E. canis DIVA antigenand the result is positive and no test is completed with an E. canisvaccine antigen, then the animal's status would be infected, butvaccination status unknown.

TABLE 1 Infection/Vaccination Status Result with E. canis Result with E.canis of Animal DIVA antigen vaccine antigen* Infected, vaccinationstatus Positive Not Done unknown Infected, vaccination status PositivePositive unknown Vaccinated, not infected Negative Positive Vaccinatedand/or infected Not Done Positive Not infected, not vaccinated NegativeNegative Not infected, vaccination Negative Not Done status unknown Notvaccinated, not infected Not Done Negative *An E. canis vaccine antigenspecifically binds an antibody that is a component of an animal's immuneresponse to a particular E. canis vaccine. An E. canis vaccine antigennot only specifically binds an antibody that is a component of ananimal's immune response to a particular E. canis vaccine, but can alsobind to antibodies that are a component of an animal's immune responseto infection by E. canis.

Another aspect of the invention provides a method for determining thepresence or absence of an antibody or antigen-binding fragment thereof,in a test sample, wherein the antibody or antigen-binding fragmentthereof specifically binds to a purified polypeptide consisting of SEQID NOs:10, 22-33 or combinations thereof. The method comprisescontacting the test sample with a purified polypeptide comprising SEQ IDNO:10, 22-33 or combinations thereof under conditions suitable forspecific binding of the purified polypeptide to the antibody orantigen-binding fragment thereof and detecting the presence or absenceof specific binding. The presence of specific binding indicates thepresence of the antibody or antigen-binding fragment thereof, and theabsence of specific binding indicates the absence the antibody orantigen-binding fragment thereof.

Vaccines may not be completely effective at preventing or amelioratinginfection. Therefore, it is desirable to have a method to determine if avaccinated animal has become infected despite the vaccination. SEQ IDNOs:10, 22-33 do not detect anti-E. canis antibodies in dogs that havebeen vaccinated for E. canis and that are not infected with E. canis.SEQ ID NOs:10, 22-33 can be used to detect E. canis infection in dogsthat have received or have not received an E. canis vaccine. In oneembodiment of the invention, the animal becomes infected with E. canisafter receiving an E. canis vaccine and detection of E. canis is stillpossible.

Another aspect of the invention comprises a composition comprising orconsisting of one or more purified polypeptides comprising or consistingof SEQ ID NOs:10, 22-33 or combinations thereof. A polypeptide of theinvention can be post-translationally modified. A purified polypeptideis a polypeptide preparation that is substantially free of cellularmaterial, other types of polypeptides, chemical precursors, chemicalsused in synthesis of the polypeptide, or combinations thereof. Apolypeptide preparation that is substantially free of cellular material,culture medium, chemical precursors, chemicals used in synthesis of thepolypeptide, etc., has less than about 30%, 20%, 10%, 5%, 1% or more ofother polypeptides, culture medium, chemical precursors, and/or otherchemicals used in synthesis. Therefore, a purified polypeptide is about70%, 80%, 90%, 95%, 99% or more pure. A purified polypeptide does notinclude unpurified or semi-purified cell extracts or mixtures ofpolypeptides that are less than 70% pure.

One embodiment of the invention provides a purified polypeptidecomprising SEQ ID NOs:22-33, wherein the polypeptide consists of lessthan about 50, 45, 40, 35, 30, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or less (or any range between 50and 6) contiguous naturally occurring Ehrlichia canis amino acids (i.e,the purified polypeptide does not encompass the entire naturallyoccurring Ehrlichia canis polypeptide). Naturally occurring Ehrlichiacanis amino acids are any polypeptides naturally produced by anEhrlichia canis organism. In one embodiment of the invention a purifiedpolypeptide comprises SEQ ID NOs:22-33, wherein the polypeptidecomprises more than about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 35, 40, 50, 60, 70, 80, 90, 100,or more contiguous naturally occurring Ehrlichia canis amino acids (orany range between about 6 and 100 amino acids).

The fact that polypeptides SEQ ID NOs:22-33 are smaller than a fulllength Ehrlichia canis polypeptide is important because smallerpolypeptides can have greater specificity and/or sensitivity than fulllength polypeptides in detection assays. Additionally, these smallerpolypeptides can be less expensive to manufacture, and may be obtainedat greater purity than the full length polypeptide.

One embodiment of the invention provides a purified polypeptide that isless than about 50, 45, 40, 35, 30, 26, 25, 24, 23, 22, 21, 20, 19, 18,17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6 contiguous naturallyEhrlichia canis amino acids and greater than about 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 35, 40,50, 60, 70, 80, 90, or 100 contiguous amino acids of SEQ ID NOs:22-33(or any range between 6 and 100 amino acids). Therefore, a polypeptideof the invention can be, for example, about 15 to about 30; about 15 toabout 50; or about 15 to about 100 amino acids in length.

Variant polypeptides are at least about 79%, 80%, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or moreidentical to the polypeptide sequences shown in SEQ ID NOs:22-33 and arealso polypeptides of the invention. Variant polypeptides have one ormore conservative amino acid variations or other minor modifications andretain biological activity, i.e., are biologically functionalequivalents. A biologically active equivalent has substantiallyequivalent function when compared to the corresponding wild-typepolypeptide. In one embodiment of the invention a polypeptide has about1, 2, 3, 4, 5, 10, 20 or less conservative amino acid substitutions.

These polypeptides may have additional amino acid residues beyond thosein SEQ ID NOs:22-33. That is, the polypeptides may have additional aminoacids residues added onto the 5′ or 3′ end of the polypeptides, whilethe sequence identity along 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 35, 40, 45, or 48 contiguous amino acids (dependingon the length of the specific SEQ ID NO) of SEQ ID NOs:22-33 is at leastabout 95%. The additional amino acids added onto the 5′ or 3′ end of thepolypeptides are not considered to affect the sequence identitypercentage. The additional amino acids can be those that naturally occuror can be non-naturally occurring amino acids. The polypeptides can be,for example, from about 15 to about 50 or about 75 amino acids inlength.

Variant polypeptides have one or more conservative amino acidsubstitutions, deletions additions or other minor modifications andretain biological activity, i.e., are biologically functionalequivalents. A biologically active equivalent has substantiallyequivalent function when compared to the corresponding wild-typepolypeptide.

Percent sequence identity has an art recognized meaning and there are anumber of methods to measure identity between two polypeptide orpolynucleotide sequences. See, e.g., Lesk, Ed., Computational MolecularBiology, Oxford University Press, New York, (1988); Smith, Ed.,Biocomputing: Informatics And Genome Projects, Academic Press, New York,(1993); Griffin & Griffin, Eds., Computer Analysis Of Sequence Data,Part I, Humana Press, New Jersey, (1994); von Heinje, Sequence AnalysisIn Molecular Biology, Academic Press, (1987); and Gribskov & Devereux,Eds., Sequence Analysis Primer, M Stockton Press, New York, (1991).Methods for aligning polynucleotides or polypeptides are codified incomputer programs, including the GCG program package (Devereux et al.,Nuc. Acids Res. 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul et al.,J. Molec. Biol. 215:403 (1990)), and Bestfit program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711) whichuses the local homology algorithm of Smith and Waterman (Adv. App.Math., 2:482-489 (1981)). For example, the computer program ALIGN whichemploys the FASTA algorithm can be used, with an affine gap search witha gap open penalty of −12 and a gap extension penalty of −2.

When using any of the sequence alignment programs to determine whether aparticular sequence is, for instance, about 95% identical to a referencesequence, the parameters are set such that the percentage of identity iscalculated over the full length of the reference polynucleotide orpolypeptide and that gaps in identity of up to 5% of the total number ofnucleotides or amino acids in the reference polynucleotide are allowed.

Variant polypeptides can generally be identified by modifying one of thepolypeptide sequences of the invention, and evaluating the properties ofthe modified polypeptide to determine if it is a biological equivalent.A variant is a biological equivalent if it reacts substantially the sameas a polypeptide of the invention in an assay such as animmunohistochemical assay, an enzyme-linked immunosorbent Assay (ELISA),a radioimmunoassay (RIA), immunoenzyme assay or a western blot assay,e.g. has 90-110% of the activity of the original polypeptide. In oneembodiment, the assay is a competition assay wherein the biologicallyequivalent polypeptide is capable of reducing binding of the polypeptideof the invention to a corresponding reactive antigen or antibody byabout 80, 95, 99, or 100%. An antibody that specifically binds acorresponding wild-type polypeptide also specifically binds the variantpolypeptide.

A conservative substitution is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. In general, the following groups of amino acids representconservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr;(2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg,his; and (5) phe, tyr, trp, his.

A polypeptide of the invention can further comprise a signal (or leader)sequence that co-translationally or post-translationally directstransfer of the protein. The polypeptide can also comprise a linker orother sequence for ease of synthesis, purification or identification ofthe polypeptide (e.g., poly-His), or to enhance binding of thepolypeptide to a solid support. For example, a polypeptide can beconjugated to an immunoglobulin Fc region or bovine serum albumin.

A polypeptide can be covalently or non-covalently linked to an aminoacid sequence to which the polypeptide is not normally associated within nature, i.e., a heterologous amino acid sequence. A heterologousamino acid sequence can be from a non-E. canis organism, a syntheticsequence, or an E. canis sequence not located at the carboxy or aminoterminus of a polypeptide of the invention in nature. Additionally, apolypeptide can be covalently or non-covalently linked to compounds ormolecules other than amino acids such as indicator reagents. Apolypeptide can be covalently or non-covalently linked to an indicatorreagent, an amino acid spacer, an amino acid linker, a signal sequence,a stop transfer sequence, a transmembrane domain, a protein purificationligand, or a combination thereof. A polypeptide can also be linked to amoiety (i.e., a functional group that can be a polypeptide or othercompound) that enhances an immune response (e.g., cytokines such asIL-2), a moiety that facilitates purification (e.g., affinity tags suchas a six-histidine tag, trpE, glutathione, maltose binding protein), ora moiety that facilitates polypeptide stability (e.g., polyethyleneglycol; amino terminus protecting groups such as acetyl, propyl,succinyl, benzyl, benzyloxycarbonyl or t-butyloxycarbonyl; carboxylterminus protecting groups such as amide, methylamide, and ethylamide).In one embodiment of the invention a protein purification ligand can beone or more C amino acid residues at, for example, the amino terminus orcarboxy terminus or both termini of a polypeptide of the invention. Anamino acid spacer is a sequence of amino acids that are not associatedwith a polypeptide of the invention in nature. An amino acid spacer cancomprise about 1, 5, 10, 20, 100, or 1,000 amino acids.

If desired, a polypeptide of the invention can be part of a fusionprotein, which can also contain other amino acid sequences, such asamino acid linkers, amino acid spacers, signal sequences, TMR stoptransfer sequences, transmembrane domains, as well as ligands useful inprotein purification, such as glutathione-S-transferase, histidine tag,and Staphylococcal protein A, or combinations thereof. More than onepolypeptide of the invention can be present in a fusion protein.Fragments of polypeptides of the invention can be present in a fusionprotein of the invention. A polypeptide of the invention can be operablylinked to non-Ehrlichia canis proteins or non-Ehrlichia canis p16proteins to form fusion proteins. A fusion protein of the invention cancomprise one or more polypeptides shown in SEQ ID NO: 2, 4, 6, 8, 10,12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33 or fragments thereof, or combinations thereof. A fusionprotein does not occur in nature. The term “operably linked” means thatthe polypeptide of the invention and the other polypeptides are fusedin-frame to each other either to the N-terminus or C-terminus of thepolypeptide of the invention.

Polypeptides of the invention can be in a multimeric form. That is, apolypeptide can comprise one or more copies of SEQ ID NOs: 2, 4, 6, 8,10, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33 or a combination thereof. A multimeric polypeptide can bea multiple antigen peptide (MAP). See e.g., Tam, J. Immunol. Methods,196:17-32 (1996).

Polypeptides of the invention can comprise an antigen that is recognizedby an antibody specific for E. canis. The antigen can comprise one ormore epitopes (i.e., antigenic determinants). An epitope can be a linearepitope, sequential epitope or a conformational epitope. Epitopes withina polypeptide of the invention can be identified by several methods.See, e.g., U.S. Pat. No. 4,554,101; Jameson & Wolf, CABIOS 4:181-186(1988). For example, a polypeptide of the invention can be isolated andscreened. A series of short peptides, which together span an entirepolypeptide sequence, can be prepared by proteolytic cleavage. Bystarting with, for example, 30-mer polypeptide fragments (or smallerfragments), each fragment can be tested for the presence of epitopesrecognized in an ELISA. For example, in an ELISA assay an E. canispolypeptide, such as a 30-mer polypeptide fragment, is attached to asolid support, such as the wells of a plastic multi-well plate. Apopulation of antibodies are labeled, added to the solid support andallowed to bind to the unlabeled antigen, under conditions wherenon-specific absorption is blocked, and any unbound antibody and otherproteins are washed away. Antibody binding is detected by, for example,a reaction that converts a colorless substrate into a colored reactionproduct. Progressively smaller and overlapping fragments can then betested from an identified 30-mer to map the epitope of interest.

In one embodiment of the invention, a DIVA antigen comprises animmunodominant epitope or region. That is, an epitope or region thatmore frequently elicits and binds to antibodies in a population thereofwhen compared with other epitopes. An antigen can have one or moreimmunodominant epitopes. Immunodominant epitopes can be mapped on, forexample, a polypeptide after the polypeptide has been administered to ananimal or prior to such administration. See e.g., U.S. Pat. Publ.2004/0209324.

A polypeptide of the invention can be produced recombinantly. Apolynucleotide encoding a polypeptide of the invention can be introducedinto a recombinant expression vector, which can be expressed in asuitable expression host cell system using techniques well known in theart. A variety of bacterial, yeast, plant, mammalian, and insectexpression systems are available in the art and any such expressionsystem can be used. Optionally, a polynucleotide encoding a polypeptidecan be translated in a cell-free translation system. A polypeptide canalso be chemically synthesized or obtained from E. canis cells.

An immunogenic polypeptide of the invention can comprise an amino acidsequence shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or fragmentsthereof. An immunogenic polypeptide can elicit antibodies or otherimmune responses (e.g., T-cell responses of the immune system) thatrecognize epitopes of a polypeptide having SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, or 33. An immunogenic polypeptide of the invention can also be afragment of a polypeptide that has an amino acid sequence shown in SEQID NOs: 2, 4, 6, 8, 10, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, or 33. An immunogenic polypeptidefragment of the invention can be about 50, 45, 40, 35, 30, 26, 25, 24,23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 orless (or any range between about 50 and about 6) amino acids in length.An immunogenic polypeptide fragment of the invention can be more thanabout 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 30, 35, 40, 50, 60, 70, 80, 90, 100, or more amino acidsin length (or any range between about 6 and about 100 amino acids).

Antibodies specific for E. canis can be detected in biological fluids ortissues by any method known in the art using the polypeptides of theinvention. The simplest methods generally are immunoassay methods. Onesuch method is a competition-based method wherein serum samples arepreincubated with an E. canis antigen that is not an element of an E.canis vaccine (e.g., an E. canis DIVA antigen), and then added to asolid phase, such a microtiter plate, having an immobilized monoclonalantibody specific for the E. canis DIVA antigen. Antibodies specific forthe E. canis DIVA antigen in the sample will prevent the E. canis DIVAantigen from binding to the immobilized antibody. Detection of anybinding of the E. canis DIVA antigen to the immobilized antibody can bedetermined by adding a second binding partner for the E. canis antigen,either directly labeled or capable of becoming labeled through bindingto another binding partner having a label. A positive sample, i.e. asample having antibodies specific for an E. canis DIVA antigen, isassociated with a decrease in signal from the label.

In one particular embodiment, antibodies to an E. canis DIVA antigen ina biological sample can be detected by contacting the sample with an E.canis DIVA antigen and adding the sample to microtiter plate coated withan anti-DIVA antigen monoclonal antibody. Binding of the DIVA antigen tothe microtiter plate can be detected by adding a rabbit polyclonalantibody against the DIVA antigen and adding an HRP-conjugated donkeyanti-rabbit polyclonal antibody. Antibodies in the sample will preventthe binding of the DIVA antigen to the immobilized antibody, therebycausing a decrease in signal.

Another method for detecting antibodies specific for an E. canis DIVAantigen is a sandwich assay where a biological sample suspected ofcontaining an antibody specific for an E. canis DIVA antigen iscontacted with an immobilized E. canis DIVA antigen to form animmunological complex. The presence of an antibody specific for an E.canis DIVA antigen is determined by the detection of the binding of alabeled binding partner for the E. canis antibody, such as a secondantibody.

In one aspect of the invention, E. canis DIVA antigens can beimmobilized on a suitable solid support. A biological sample is broughtinto contact with the E. canis DIVA antigen, to which the anti-E. canisantibodies bind, if such antibodies are present in the sample. Thebinding can be detected by any suitable means, e.g., enzymes,radionuclides, particulates or fluorescent labels. In a suitableembodiment, the detection reagent can be associated with a protein thatis the same or similar to that which is used to capture anti-E. canisantibodies (if present). In one particular embodiment, antibodies to E.canis can be detected by immobilizing an E. canis antigen on a solidsupport. Biological samples can be contacted with the solid support and,following the removal of unbound sample, binding of the E. canisantibodies to the antigen can be accomplished with, for example, alabeled IgG antibody.

DIVA antigens of the invention can also comprise mimitopes of DIVAantigens of the invention. A mimitope is a random peptide epitope thatmimics a natural antigenic epitope during epitope presentation. Randompeptide epitopes can be identified by generating or selecting a libraryof random peptide epitopes. The library is contacted with an antibody.Mimitopes are identified that are specifically immunoreactive with theantibody. Random peptide libraries can, for example, be displayed onphage or generated as combinatorial libraries.

E. canis DIVA antigens, e.g., polypeptides, can be natural, i.e.,isolated from a natural source, or can be synthetic (i.e., chemicallysynthesized or recombinantly produced using genetic engineeringtechniques). Natural proteins can be isolated from the whole bacteriumby conventional techniques, such as affinity chromatography. Polyclonalor monoclonal antibodies can be used to prepare a suitable affinitycolumn by well-known techniques.

Proteins that are immunologically cross-reactive with a natural E. canisprotein can be chemically synthesized. For example, polypeptides havingfewer than about 100 amino acids, more usually fewer than about 80 aminoacids, and typically fewer than about 50 amino acids, can be synthesizedby the well-known Merrifield solid-phase synthesis method where aminoacids are sequentially added to a growing chain. Merrifield, 1963, J.Am. Chem. Soc., 85:2149-2156). Recombinant proteins can also be used.These proteins can be produced by expression in cultured cells ofrecombinant DNA molecules encoding a desired portion of the E. canisgenome. The portion of the E. canis genome can itself be natural orsynthetic, with natural genes obtainable from the isolated bacterium byconventional techniques.

E. canis Polynucleotides

Polynucleotides of the invention contain less than an entire microbialgenome and can be single- or double-stranded nucleic acids. Apolynucleotide can be RNA, DNA, cDNA, genomic DNA, chemicallysynthesized RNA or DNA or combinations thereof. The polynucleotides canbe purified free of other components, such as proteins, lipids and otherpolynucleotides. For example, the polynucleotide can be 50%, 75%, 90%,95%, 96%, 97%, 98%, 99%, or 100% purified. A nucleic acid moleculeexisting among hundreds to millions of other nucleic acid moleculeswithin, for example, cDNA or genomic libraries, or gel slices containinga genomic DNA restriction digest are not to be considered an isolatedpolynucleotide. The polynucleotides of the invention encode thepolypeptides of the invention described above. In one embodiment of theinvention the polynucleotides encode polypeptides shown in SEQ ID NOs:2,4, 6, 8, 10, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, fragments thereof, or combinations thereof.Polynucleotides of the invention can consist of less than about 200,120, 100, 90, 75, 60, 57, 54, 45 (or any range between 200 and 45)contiguous, naturally occurring Ehrlichia canis polynucleotides.Polynucleotides of the invention can consist of greater than about 45,54, 57, 60, 75, 90, 100, 120, 150, 200, (or any range between 45 and200), or more contiguous, naturally occurring Ehrlichia canispolynucleotides. The purified polynucleotides can comprise additionalheterologous nucleotides (that is, nucleotides that are not fromEhrlichia canis) and even additional Ehrlichia canis amino acids as longas they do not naturally occur contiguously with Ehrlichia canis p16polynucleotides or other polynucleotides of the invention.Polynucleotides of the invention can comprise other nucleotidesequences, such as sequences coding for linkers, signal sequences, TMRstop transfer sequences, transmembrane domains, or ligands useful inprotein purification such as glutathione-S-transferase, histidine tag,and Staphylococcal protein A.

Polynucleotides of the invention can be isolated. An isolatedpolynucleotide is a naturally-occurring polynucleotide that is notimmediately contiguous with one or both of the 5′ and 3′ flankinggenomic sequences that it is naturally associated with. An isolatedpolynucleotide can be, for example, a recombinant DNA molecule of anylength, provided that the nucleic acid sequences naturally foundimmediately flanking the recombinant DNA molecule in anaturally-occurring genome is removed or absent. Isolatedpolynucleotides also include non-naturally occurring nucleic acidmolecules. A nucleic acid molecule existing among hundreds to millionsof other nucleic acid molecules within, for example, cDNA or genomiclibraries, or gel slices containing a genomic DNA restriction digest arenot to be considered an isolated polynucleotide. The complete nucleotidesequence for E. canis is available from, e.g., GenBank as accessionnumber NCBI: NZ_AAEJ01000001.

Polynucleotides of the invention can also comprise fragments that encodeimmunogenic polypeptides. Polynucleotides of the invention can encodefull-length polypeptides, polypeptide fragments, and variant or fusionpolypeptides.

Degenerate nucleotide sequences encoding polypeptides of the invention,as well as homologous nucleotide sequences that are at least about 80,or about 90, 96, 98, or 99% identical to the polynucleotide sequences ofthe invention and the complements thereof are also polynucleotides ofthe invention. Percent sequence identity can be calculated as describedin the “Polypeptides” section. Degenerate nucleotide sequences arepolynucleotides that encode a polypeptide of the invention or fragmentsthereof, but differ in nucleic acid sequence from the wild-typepolynucleotide sequence, due to the degeneracy of the genetic code.Complementary DNA (cDNA) molecules, species homologs, and variants of E.canis polynucleotides that encode biologically functional E. canispolypeptides also are E. canis polynucleotides. Polynucleotides of theinvention can be isolated from nucleic acid sequences present in, forexample, a biological sample, such as blood, serum, saliva, or tissuefrom an infected individual. Polynucleotides can also be synthesized inthe laboratory, for example, using an automatic synthesizer. Anamplification method such as PCR can be used to amplify polynucleotidesfrom either genomic DNA or cDNA encoding the polypeptides.

Polynucleotides of the invention can comprise coding sequences fornaturally occurring polypeptides or can encode altered sequences that donot occur in nature. If desired, polynucleotides can be cloned into anexpression vector comprising expression control elements, including forexample, origins of replication, promoters, enhancers, or otherregulatory elements that drive expression of the polynucleotides of theinvention in host cells. An expression vector can be, for example, aplasmid, such as pBR322, pUC, or ColE1, or an adenovirus vector, such asan adenovirus Type 2 vector or Type 5 vector. Optionally, other vectorscan be used, including but not limited to Sindbis virus, simian virus40, alphavirus vectors, poxvirus vectors, and cytomegalovirus andretroviral vectors, such as murine sarcoma virus, mouse mammary tumorvirus, Moloney murine leukemia virus, and Rous sarcoma virus.Minichromosomes such as MC and MC1, bacteriophages, phagemids, yeastartificial chromosomes, bacterial artificial chromosomes, virusparticles, virus-like particles, cosmids (plasmids into which phagelambda cos sites have been inserted) and replicons (genetic elementsthat are capable of replication under their own control in a cell) canalso be used.

Methods for preparing polynucleotides operably linked to an expressioncontrol sequence and expressing them in a host cell are well-known inthe art. See, e.g., U.S. Pat. No. 4,366,246. A polynucleotide of theinvention is operably linked when it is positioned adjacent to or closeto one or more expression control elements, which direct transcriptionand/or translation of the polynucleotide.

Polynucleotides of the invention can be used, for example, as probes orprimers, for example PCR primers, to detect the presence of E. canispolynucleotides in a test sample, such as a biological sample. Probesare molecules capable of interacting with a target nucleic acid,typically in a sequence specific manner, for example, throughhybridization. Primers are a subset of probes that can support anenzymatic manipulation and that can hybridize with a target nucleic acidsuch that the enzymatic manipulation occurs. A primer can be made fromany combination of nucleotides or nucleotide derivatives or analogsavailable in the art that do not interfere with the enzymaticmanipulation.

A probe or primer can be about 10, 15, 20, 25, 30, 40, 50, 60, 70, 80,90, 100 or more contiguous nucleotides that encode polypeptides shownin, e.g., SEQ ID NOs:22-33.

The hybridization of nucleic acids is well understood in the art.Typically a probe can be made from any combination of nucleotides ornucleotide derivatives or analogs available in the art. The ability ofsuch probes and primers to specifically hybridize to E. canispolynucleotide sequences will enable them to be of use in detecting thepresence of complementary sequences in a given test sample.Polynucleotide probes and primers of the invention can hybridize tocomplementary sequences in a test sample such as a biological sample,including saliva, sputum, blood, plasma, serum, urine, feces,cerebrospinal fluid, amniotic fluid, wound exudate, or tissue.Polynucleotides from the sample can be, for example, subjected to gelelectrophoresis or other size separation techniques or can beimmobilized without size separation. The polynucleotide probes orprimers can be labeled. Suitable labels, and methods for labeling probesand primers are known in the art, and include, for example, radioactivelabels incorporated by nick translation or by kinase, biotin labels,fluorescent labels, chemiluminescent labels, bioluminescent labels,metal chelator labels and enzyme labels. The polynucleotides from thesample are contacted with the probes or primers under hybridizationconditions of suitable stringencies.

Depending on the application, varying conditions of hybridization can beused to achieve varying degrees of selectivity of the probe or primertowards the target sequence. For applications requiring highselectivity, relatively stringent conditions can be used, such as lowsalt and/or high temperature conditions, such as provided by a saltconcentration of from about 0.02 M to about 0.15 M salt at temperaturesof from about 50° C. to about 70° C. For applications requiring lessselectivity, less stringent hybridization conditions can be used. Forexample, salt conditions from about 0.14 M to about 0.9M salt, attemperatures ranging from about 20° C. to about 55° C. The presence of ahybridized complex comprising the probe or primer and a complementarypolynucleotide from the test sample indicates the presence of E. canisor an E. canis polynucleotide in the sample.

Antibodies

Antibodies of the invention are antibody molecules that specificallybind to an E. canis polypeptide of the invention, variant polypeptidesof the invention, or fragments thereof. An antibody of the invention canbe specific for an Ehrlichia canis polypeptide, for example, an antibodyspecific for one or more of SEQ ID NOs:10, 22-33. An antibody of theinvention can be a polyclonal antibody, a monoclonal antibody, a singlechain antibody (scFv), or an antigen-binding fragment of an antibody.Antigen binding fragments of antibodies are the antigen-binding portionof an intact antibody comprising the antigen binding site or variableregion of an intact antibody, wherein the portion is free of theconstant heavy chain domains of the Fc region of the intact antibody.Examples of antigen binding fragments include Fab, Fab′, Fab′-SH,F(ab′)₂ and F_(v) fragments.

An antibody of the invention can be any antibody class, including forexample, IgG, IgM, IgA, IgD and IgE. An antibody or fragment thereofbinds to an epitope of a polypeptide of the invention. An antibody canbe made in vivo in suitable laboratory animals or in vitro usingrecombinant DNA techniques. Means for preparing and characterizingantibodies are well know in the art. See, e.g., Dean, Methods Mol. Biol.80:23-37 (1998); Dean, Methods Mol. Biol. 32:361-79 (1994); Baileg,Methods Mol. Biol. 32:381-88 (1994); Gullick, Methods Mol. Biol.32:389-99 (1994); Drenckhahn et al. Methods Cell. Biol. 37:7-56 (1993);Morrison, Ann. Rev. Immunol. 10:239-65 (1992); Wright et al. Crit. Rev.Immunol. 12:125-68 (1992). For example, polyclonal antibodies can beproduced by administering a polypeptide of the invention to an animal,such as a human or other primate, mouse, rat, rabbit, guinea pig, goat,pig, dog, cow, sheep, donkey, or horse. Serum from the immunized animalis collected and the antibodies are purified from the plasma by, forexample, precipitation with ammonium sulfate, followed bychromatography, such as affinity chromatography. Techniques forproducing and processing polyclonal antibodies are known in the art.

“Specifically binds” or “specific for” means that a first antigen, e.g.,an E. canis polypeptide, recognizes and binds to an antibody of theinvention with greater affinity than to other, non-specific molecules.“Specifically binds” or “specific for” also means a first antibody,e.g., an antibody raised against SEQ ID NOs:22-33, recognizes and bindsto SEQ ID NOs:22-33, with greater affinity than to other non-specificmolecules. A non-specific molecule is an antigen that shares no commonepitope with the first antigen. In a preferred embodiment of theinvention a non-specific molecule is not derived from Ehrlichia sp., andin particular is not derived from Ehrlichia chaffeensis or Ehrlichiacanis. “Ehrlichia sp.” refers to all species of the genus Ehrlichia. Forexample, an antibody raised against a first antigen (e.g., apolypeptide) to which it binds more efficiently than to a non-specificantigen can be described as specifically binding to the first antigen.In one embodiment, an antibody or antigen-binding portion thereof of theinvention specifically binds to a polypeptide of SEQ ID NOs:2, 4, 6, 8,10, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or fragments thereof when it binds with a binding affinity K_(a) of 10⁷l/mol or more. Specific binding can be tested using, for example, anenzyme-linked immunosorbant assay (ELISA), a radioimmunoassay (RIA), ora western blot assay using methodology well known in the art.

Antibodies of the invention include antibodies and antigen bindingfragments thereof that (a) compete with a reference antibody for bindingto SEQ ID NOs:22-33 or antigen binding fragments thereof; (b) binds tothe same epitope of SEQ ID NOs:22-33 or antigen binding fragmentsthereof as a reference antibody; (c) binds to SEQ ID NOs:22-33 orantigen binding fragments thereof with substantially the same K_(d) as areference antibody; and/or (d) binds to SEQ ID NOs:22-33 or fragmentsthereof with substantially the same off rate as a reference antibody,wherein the reference antibody is an antibody or antigen-bindingfragment thereof that specifically binds to a polypeptide of SEQ IDNOs:22-33 or antigen binding fragments thereof with a binding affinityK_(a) of 10⁷ l/mol or more.

Additionally, monoclonal antibodies directed against epitopes present ona polypeptide of the invention can also be readily produced. Forexample, normal B cells from a mammal, such as a mouse, which wasimmunized with a polypeptide of the invention can be fused with, forexample, HAT-sensitive mouse myeloma cells to produce hybridomas.Hybridomas producing E. canis-specific antibodies can be identifiedusing RIA or ELISA and isolated by cloning in semi-solid agar or bylimiting dilution. Clones producing E. canis-specific antibodies areisolated by another round of screening. Monoclonal antibodies can bescreened for specificity using standard techniques, for example, bybinding a polypeptide of the invention to a microtiter plate andmeasuring binding of the monoclonal antibody by an ELISA assay.Techniques for producing and processing monoclonal antibodies are knownin the art. See e.g., Kohler & Milstein, Nature, 256:495 (1975).Particular isotypes of a monoclonal antibody can be prepared directly,by selecting from the initial fusion, or prepared secondarily, from aparental hybridoma secreting a monoclonal antibody of a differentisotype by using a sib selection technique to isolate class-switchvariants. See Steplewski et al., P.N.A.S. U.S.A. 82:8653 1985; Spria etal., J. Immunolog. Meth. 74:307, 1984. Monoclonal antibodies of theinvention can also be recombinant monoclonal antibodies. See, e.g., U.S.Pat. No. 4,474,893; 4,816,567. Antibodies of the invention can also bechemically constructed. See, e.g., U.S. Pat. No. 4,676,980.

Antibodies of the invention can be chimeric (see, e.g., U.S. Pat. No.5,482,856), humanized (see, e.g., Jones et al., Nature 321:522 (1986);Reichmann et al., Nature 332:323 (1988); Presta, Curr. Op. Struct. Biol.2:593 (1992)), or human antibodies. Human antibodies can be made by, forexample, direct immortilization, phage display, transgenic mice, or aTrimera methodology, see e.g., Reisener et al., Trends Biotechnol.16:242-246 (1998).

Antibodies that specifically bind E. canis antigens (e.g., E. canispolypeptides shown in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33), areparticularly useful for detecting the presence of E. canis or E. canisantigens in a sample, such as a serum, blood, plasma, fecal, cell,tissue, urine or saliva sample from an E. canis-infected animal such asa human or dog. An immunoassay for E. canis or an E. canis antigen canutilize one antibody or several antibodies. An immunoassay for E. canisor an E. canis antigen can use, for example, a monoclonal antibodyspecific for an E. canis epitope, a combination of monoclonal antibodiesspecific for epitopes of one E. canis polypeptide, monoclonal antibodiesspecific for epitopes of different E. canis polypeptides, polyclonalantibodies specific for the same E. canis antigen, polyclonal antibodiesspecific for different E. canis antigens, or a combination of monoclonaland polyclonal antibodies. Immunoassay protocols can be based upon, forexample, competition, direct reaction, or sandwich type assays using,for example, labeled antibody. Antibodies of the invention can belabeled with any type of label known in the art, including, for example,fluorescent, chemiluminescent, radioactive, enzyme, colloidal metal,radioisotope and bioluminescent labels.

Antibodies of the invention or antigen-binding fragments thereof can bebound to a support and used to detect the presence of E. canis or an E.canis antigen, e.g., an E. canis DIVA antigen or E. canis vaccineantigen. Supports include, for example, glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, agaroses and magletite.

Antibodies of the invention can further be used to isolate E. canisorganisms or E. canis antigens by immunoaffinity columns. The antibodiescan be affixed to a solid support by, for example, adsorbtion or bycovalent linkage so that the antibodies retain their immunoselectiveactivity. Optionally, spacer groups can be included so that the antigenbinding site of the antibody remains accessible. The immobilizedantibodies can then be used to bind E. canis organisms or E. canisantigens from a sample, such as a biological sample including saliva,serum, sputum, blood, urine, feces, cerebrospinal fluid, amniotic fluid,wound exudate, or tissue. The bound E. canis organisms or E. canisantigens are recovered from the column matrix by, for example, a changein pH.

Antibodies of the invention can also be used in immunolocalizationstudies to analyze the presence and distribution of a polypeptide of theinvention during various cellular events or physiological conditions.Antibodies can also be used to identify molecules involved in passiveimmunization and to identify molecules involved in the biosynthesis ofnon-protein antigens. Identification of such molecules can be useful invaccine development. Antibodies of the invention, including, forexample, monoclonal antibodies and single chain antibodies, can be usedto monitor the course of amelioration of a disease caused by E. canis.By measuring the increase or decrease of E. canis antibodies specificfor E. canis antigens in a test sample from an animal, it can bedetermined whether a particular therapeutic regiment aimed atameliorating the disorder is effective. Antibodies can be detectedand/or quantified using for example, direct binding assays such as RIA,ELISA, or western blot assays.

Detection

The methods of the invention can be used to detect antibodies orantigen-binding antibody fragments specific for Ehrlichia canis antigensor Ehrlichia canis polynucleotides in a test sample, such as abiological sample, an environmental sample, or a laboratory sample. Atest sample can potentially comprise Ehrlichia sp. polynucleotides,Ehrlichia canis polynucleotides, Ehrlichia sp. polypeptides, Ehrlichiacanis polypeptides, antibodies specific for Ehrlichia sp., and/orantibodies specific for Ehrlichia canis, unrelated polynucleotide andpolypeptides, combinations thereof, or none of the above. A biologicalsample can include, for example, sera, blood, cells, plasma, saliva,urine, feces, or tissue from a mammal such as a horse, cat, dog orhuman. The test sample can be untreated, precipitated, fractionated,separated, diluted, concentrated, or purified.

In one embodiment methods of the invention comprise contacting one ormore polypeptides of the invention with a test sample under conditionsthat allow polypeptide/antibody complexes, i.e., immunocomplexes, toform. That is, polypeptides of the invention specifically bind toantibodies specific for Ehrlichia canis antigens located in the sample.In one embodiment of the invention one or more polypeptides of theinvention specifically bind to antibodies that are specific forEhrlichia canis antigens and do not specifically bind to antigens fromother pathogens, such as, e.g., Ehrlichia chaffeensis antigens. One ofskill in the art is familiar with assays and conditions that are used todetect antibody/polypeptide complex binding. The formation of a complexbetween polypeptides and antibodies in the sample is detected. Theformation of antibody/polypeptide complexes is an indication thatEhrlichia canis polypeptides are present in the sample. The lack ofdetection of the polypeptide/antibody complexes is an indication thatEhrlichia canis polypeptides are not present in the sample.

Antibodies of the invention can be used in a method of detection ofEhrlichia canis antigens by obtaining a test sample from, e.g., a humanor animal suspected of having an Ehrlichia canis infection. The testsample is contacted with antibodies of the invention under conditionsenabling the formation of antibody-antigen complexes (i.e.,immunocomplexes). One of skill in the art is aware of conditions thatenable and are appropriate for formation of antigen/antibody complexes.The amount of antibody-antigen complexes can be determined bymethodology known in the art. A level that is higher than that formed ina negative control sample indicates presence of Ehrlichia canisantigens. A negative control sample is a sample that does not compriseany Ehrlichia canis polypeptides. In one embodiment of the invention thenegative control contains no Ehrlichia sp. polypeptides. In oneembodiment of the invention an antibody is specific for Ehrlichia canisantigens and is not specific for antigens from other pathogens, such as,e.g., Ehrlichia chaffeensis antigens. Alternatively, a polypeptide ofthe invention can be contacted with a test sample. Antibodies specificfor Ehrlichia canis in a positive test sample will form antigen-antibodycomplexes under suitable conditions. The amount of antibody-antigencomplexes can be determined by methods known in the art.

In one embodiment of the invention, Ehrlichia canis infection can bedetected in a subject. A biological sample is obtained from the subject.One or more purified polypeptides comprising SEQ ID NOs:22-33 or otherpolypeptides of the invention are contacted with the biological sampleunder conditions that allow polypeptide/antibody complexes to form. Thepolypeptide/antibody complexes are detected. The detection of thepolypeptide/antibody complexes is an indication that antibodies specificfor Ehrlichia canis are present. The lack of detection of thepolypeptide/antibody complexes is an indication that the mammal does nothave antibodies specific for Ehrlichia canis.

In one embodiment of the invention the Ehrlichia canis antibodiesdetected by an immunoassay of the invention are IgG, IgM, IgA, IgD orIgE. In another embodiment of the invention, the antibodies used todetect Ehrlichia canis antigens are IgG, IgM, IgA, IgD or IgE.

In one embodiment of the invention, Ehrlichia canis infection can bedetected in a subject by about 5 days, 6 days, 7 days, 8 days, 9 days,10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days,18 days, 19 days, 20 days, 21 days or more after the subject acquiredthe Ehrlichia canis infection. In one embodiment of the invention,Ehrlichia canis infection can be detected in a subject by about 21 days,20 days, 19 days, 18 days, 17 days, 16 days, 15 days, 14 days, 13 days,12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, orless after the subject acquired the Ehrlichia canis infection.

In one embodiment of the invention, the polypeptide/antibody complex isdetected when an indicator reagent, such as an enzyme conjugate, whichis bound to the antibody, catalyzes a detectable reaction. Optionally,an indicator reagent comprising a signal generating compound can beapplied to the polypeptide/antibody complex under conditions that allowformation of a polypeptide/antibody/indicator complex. Thepolypeptide/antibody/indicator complex is detected. Optionally, thepolypeptide or antibody can be labeled with an indicator reagent priorto the formation of a polypeptide/antibody complex. The method canoptionally comprise a positive or negative control.

In one embodiment of the invention, one or more antibodies of theinvention are attached to a solid phase or substrate. A test samplepotentially comprising a protein comprising a polypeptide of theinvention is added to the substrate. One or more antibodies thatspecifically bind polypeptides of the invention are added. Theantibodies can be the same antibodies used on the solid phase or can befrom a different source or species and can be linked to an indicatorreagent, such as an enzyme conjugate. Wash steps can be performed priorto each addition. A chromophore or enzyme substrate is added and coloris allowed to develop. The color reaction is stopped and the color canbe quantified using, for example, a spectrophotometer.

In another embodiment of the invention, one or more antibodies of theinvention are attached to a solid phase or substrate. A test samplepotentially comprising a protein comprising a polypeptide of theinvention is added to the substrate. Second anti-species antibodies thatspecifically bind polypeptides of the invention are added. These secondantibodies are from a different species than the solid phase antibodies.Third anti-species antibodies are added that specifically bind thesecond antibodies and that do not specifically bind the solid phaseantibodies are added. The third antibodies can comprise an indicatorreagent such as an enzyme conjugate. Wash steps can be performed priorto each addition. A chromophore or enzyme substrate is added and coloris allowed to develop. The color reaction is stopped and the color canbe quantified using, for example, a spectrophotometer.

Assays of the invention include, but are not limited to those based oncompetition, direct reaction or sandwich-type assays, including, but notlimited to enzyme linked immunosorbent assay (ELISA), western blot, IFA,radioimmunoassay (RIA), hemagglutination (HA), fluorescence polarizationimmunoassay (FPIA), and microtiter plate assays (any assay done in oneor more wells of a microtiter plate). One assay of the inventioncomprises a reversible flow chromatographic binding assay, for example aSNAP® assay (reversible flow chromatographic assay). See e.g., U.S. Pat.No. 5,726,010.

Assays can use solid phases or substrates or can be performed byimmunoprecipitation or any other methods that do not utilize solidphases. Where a solid phase or substrate is used, one or morepolypeptides of the invention are directly or indirectly attached to asolid support or a substrate such as a microtiter well, magnetic bead,non-magnetic bead, column, matrix, membrane, fibrous mat composed ofsynthetic or natural fibers (e.g., glass or cellulose-based materials orthermoplastic polymers, such as, polyethylene, polypropylene, orpolyester), sintered structure composed of particulate materials (e.g.,glass or various thermoplastic polymers), or cast membrane film composedof nitrocellulose, nylon, polysulfone or the like (generally syntheticin nature). In one embodiment of the invention a substrate is sintered,fine particles of polyethylene, commonly known as porous polyethylene,for example, 10-15 micron porous polyethylene from Chromex Corporation(Albuquerque, N. Mex.). All of these substrate materials can be used insuitable shapes, such as films, sheets, or plates, or they may be coatedonto or bonded or laminated to appropriate inert carriers, such aspaper, glass, plastic films, or fabrics. Suitable methods forimmobilizing peptides on solid phases include ionic, hydrophobic,covalent interactions and the like. Immobilization of one or moreanalyte capture reagents, e.g., E. canis polypeptides, onto a device orsolid support is performed so that an analyte capture reagent will notbe washed away by the sample, diluent and/or wash procedures. One ormore analyte capture reagents can be attached to a surface by physicaladsorption (i.e., without the use of chemical linkers) or by chemicalbinding (i.e., with the use of chemical linkers). Chemical binding cangenerate stronger attachment of capture reagents on a surface andprovide defined orientation and conformation of the surface-boundmolecules.

In one type of assay format, one or more polypeptides can be coated on asolid phase or substrate. A test sample suspected of containinganti-Ehrlichia canis antibodies or antigen-binding fragments thereof isincubated with an indicator reagent comprising a signal generatingcompound conjugated to an antibodies or antibody fragments specific forEhrlichia canis for a time and under conditions sufficient to formantigen/antibody complexes of either antibodies of the test sample tothe polypeptides of the solid phase or the indicator reagent compoundconjugated to an antibody specific for Ehrlichia canis to thepolypeptides of the solid phase. The reduction in binding of theindicator reagent conjugated to anti-Ehrlichia canis antibodies to thesolid phase can be quantitatively measured. A measurable reduction inthe signal compared to the signal generated from, e.g., a confirmednegative Ehrlichia canis test sample indicates the presence ofanti-Ehrlichia canis antibodies in the test sample. This type of assaycan quantitate the amount of anti-Ehrlichia canis antibodies in a testsample.

In another type of assay format, one or more polypeptides of theinvention are coated onto a support or substrate. A polypeptide of theinvention is conjugated to an indicator reagent and added to a testsample. This mixture is applied to the support or substrate. Ifantibodies specific for Ehrlichia canis are present in the test samplethey will bind the one or more polypeptides conjugated to an indicatorreagent and to the one or more polypeptides immobilized on the support.The polypeptide/antibody/indicator complex can then be detected. Thistype of assay can quantitate the amount of anti-Ehrlichia canisantibodies in a test sample.

In another type of assay format, one or more polypeptides of theinvention are coated onto a support or substrate. The test sample isapplied to the support or substrate and incubated. Unbound componentsfrom the sample are washed away by washing the solid support with a washsolution. If Ehrlichia canis specific antibodies are present in the testsample, they will bind to the polypeptide coated on the solid phase.This polypeptide/antibody complex can be detected using a secondspecies-specific antibody that is conjugated to an indicator reagent.The polypeptide/antibody/anti-species antibody indicator complex canthen be detected. This type of assay can quantitate the amount ofanti-Ehrlichia canis antibodies in a test sample.

Another embodiment of the invention provides a device that is suitablefor a lateral flow assay. For example, a test sample is added to a flowmatrix at a first region (a sample application zone). The test sample iscarried in a fluid flow path by capillary action to a second region ofthe flow matrix where a label capable of binding and forming a firstcomplex with an analyte in the test sample. The first complex is carriedto a third region of the flow matrix where an E. canis polypeptide isimmobilized at a distinct location. A second complex is formed betweenan immobilized polypeptide and the first complex including the antibodyfrom the sample. For example, a first complex comprising a gold solparticle and an E. canis polypeptide bound to an E. canis antibody willspecifically bind and form a second complex with a second immobilized E.canis polypeptide or with a second antibody directed to E. canisantibodies. The label that is part of the second complex can be directlyvisualized.

In another aspect, the invention includes one or more labeled specificbinding reagents that can be mixed with a test sample prior toapplication to a device of the invention. In this case it is notnecessary to have labeled specific binding reagents deposited and driedon a specific binding reagent pad in the device. A labeled specificbinding reagent, whether added to a test sample or pre-deposited on thedevice, can be for example, a labeled antibody that specifically bindsan antibody for E. canis.

An E. canis DIVA antigen or an E. canis vaccine antigen, e.g., apolypeptide, can be an immobilized analyte capture reagent in a reactionzone (solid phase). A second analyte capture reagent, e.g. an anti-IgGor anti-IgM antibody, that has been conjugated to a label, can either beadded to the sample before the sample is added to the device, or thesecond analyte capture reagent can be incorporated into the device. Forexample the labeled specific binding reagent can be deposited and driedon a fluid flow path that provides fluid communication between thesample application zone and the solid phase. Contact of the labeledspecific binding reagent with the fluid sample results in dissolution ofthe labeled specific binging reagent.

The device can also include a liquid reagent that transports unboundmaterial (e.g., unreacted fluid sample and unbound specific bindingreagents) away from the reaction zone (solid phase). A liquid reagentcan be a wash reagent and serve only to remove unbound material from thereaction zone, or it can include a detector reagent and serve to bothremove unbound material and facilitate analyte detection. For example,in the case of a specific binding reagent conjugated to an enzyme, thedetector reagent includes a substrate that produces a detectable signalupon reaction with the enzyme-antibody conjugate at the reactive zone.In the case of a labeled specific binding reagent conjugated to aradioactive, fluorescent, or light-absorbing molecule, the detectorreagent acts merely as a wash solution facilitating detection of complexformation at the reactive zone by washing away unbound labeled reagent.

Two or more liquid reagents can be present in a device, for example, adevice can comprise a liquid reagent that acts as a wash reagent and aliquid reagent that acts as a detector reagent and facilitates analytedetection.

A liquid reagent can further include a limited quantity of an“inhibitor”, i.e., a substance that blocks the development of thedetectable end product. A limited quantity is an amount of inhibitorsufficient to block end product development until most or all excess,unbound material is transported away from the second region, at whichtime detectable end product is produced.

The formation of a polypeptide/antibody complex or apolypeptide/antibody/indicator complex can be detected by, for example,radiometric, colorimetric, fluorometric, size-separation, orprecipitation methods. Optionally, detection of a polypeptide/antibodycomplex is by the addition of a secondary antibody that is coupled to anindicator reagent comprising a signal generating compound. Indicatorreagents comprising signal generating compounds (labels) associated witha polypeptide/antibody complex can be detected using the methodsdescribed above and include chromogenic agents, catalysts such as enzymeconjugates fluorescent compounds such as fluorescein and rhodamine,chemiluminescent compounds such as dioxetanes, acridiniums,phenanthridiniums, ruthenium, and luminol, radioactive elements, directvisual labels, as well as cofactors, inhibitors, magnetic particles, andthe like. Examples of enzyme conjugates include alkaline phosphatase,horseradish peroxidase, beta-galactosidase, and the like. The selectionof a particular label is not critical, but it will be capable ofproducing a signal either by itself or in conjunction with one or moreadditional substances.

Formation of the complex is indicative of the presence of anti-Ehrlichiacanis antibodies in a test sample. Therefore, the methods of theinvention can be used to diagnose Ehrlichia canis infection in ananimal.

The methods of the invention can also indicate the amount or quantity ofanti-Ehrlichia canis antibodies in a test sample. With many indicatorreagents, such as enzyme conjugates, the amount of antibody present isproportional to the signal generated. Depending upon the type of testsample, it can be diluted with a suitable buffer reagent, concentrated,or contacted with a solid phase without any manipulation. For example,it usually is preferred to test serum or plasma samples that previouslyhave been diluted, or concentrated specimens such as urine, in order todetermine the presence and/or amount of antibody present.

The invention further comprises assay kits (e.g., articles ofmanufacture) for detecting anti-Ehrlichia canis antibodies orantigen-binding antibody fragments, or Ehrlichia canis polypeptides in asample. A kit comprises one or more polypeptides of the invention andmeans for determining binding of the polypeptide to anti-Ehrlichia canisantibodies or antibody fragments in the sample. A kit or article ofmanufacture can also comprise one or more antibodies or antibodyfragments of the invention and means for determining binding of theantibodies or antibody fragments to Ehrlichia canis polypeptides in thesample. A kit can comprise a device containing one or more polypeptidesor antibodies of the invention and instructions for use of the one ormore polypeptides or antibodies for, e.g., the identification of anEhrlichia canis infection in a mammal. The kit can also comprisepackaging material comprising a label that indicates that the one ormore polypeptides or antibodies of the kit can be used for theidentification of Ehrlichia canis infection. Other components such asbuffers, stabilizers, positive controls, negative controls, detectorreagents, and the like, known to those of ordinary skill in art, can beincluded in such test kits. The polypeptides, antibodies, assays, andkits of the invention are useful, for example, in the diagnosis ofindividual cases of Ehrlichia canis infection in a patient, as well asepidemiological studies of Ehrlichia canis outbreaks. The relativeamounts of the various reagents can be varied, to provide forconcentrations in solution of the reagents that substantially optimizethe sensitivity of the assay. Particularly, the reagents can be providedas dry powders, usually lyophilized, which on dissolution will providefor a reagent solution having the appropriate concentrations forcombining with a sample.

Polypeptides and assays of the invention can be combined with otherpolypeptides or assays to detect the presence of Ehrlichia canis alongwith other organisms. For example, polypeptides and assays of theinvention can be combined with reagents that detect heartworm and/orBorrelia burgdorferi and/or Ehrlichia chaffeensis and/or Anaplasmaplatys and/or Anaplasma phagocytophilum.

Polynucleotides of the invention can be used to detect the presence ofEhrlichia canis polynucleotides in a sample. The polynucleotides can beused to detect Ehrlichia canis polynucleotides in a sample by a simplehybridization reaction and can also be used in, e.g., polymerase chainreactions (PCR) such as a real-time PCR reaction. Methods andcompositions of the invention can also be used to differentially detectthe presence Ehrlichia canis from other Ehrlichia sp., such as Ehrlichiachaffeensis.

PCR assays are well described in the art, including, for example, U.S.Pat. Nos. 4,683,195; 4,683,202; 4,965,188. Generally, polynucleotideprimers are annealed to denatured strands of a target nucleic acid.Primer extension products are formed by polymerization ofdeoxynucleoside triphosphates by a polymerase. PCR then involvesrepetitive cycles of template nucleic acid denaturation, primerannealing and extension of the annealed primers by the action of athermostable polymerase. The process results in exponentialamplification of the target Ehrlichia canis nucleic acids in the testsample, which allows for the detection of target polynucleotidesexisting in very low concentrations in a sample.

Real-time PCR assays are based on the detection of a signal, e.g., afluorescent reporter signal. This signal increases in direct proportionto the amount of PCR product in a reaction. Real-time PCR is anyamplification technique that makes it possible to monitor the evolutionof an ongoing amplification reaction. See, Quantitation of DNA/RNA UsingReal-Time PCR Detection, Perkin Elmer Applied Biosystems (1999); PCRProtocols (Academic Press New York, 1989). By recording the amount offluorescence emission at each cycle, it is possible to monitor the PCRreaction during exponential phase where the first significant increasein the amount of PCR product correlates to the initial amount of targettemplate. The higher the starting copy number of the nucleic acidtarget, the sooner a significant increase in fluorescence is observed.

One embodiment of the invention provides a method for detecting and/orquantifying Ehrlichia canis polynucleotides in a test sample. Senseprimers and antisense primers can be added to a test sample underconditions suitable for a polymerase chain reaction. The primershybridize with Ehrlichia canis polynucleotides such that anamplification product is formed if Ehrlichia canis polynucleotides arepresent in the test sample. Amplification products are detected and thepresence and/or quantity of Ehrlichia canis polynucleotides aredetermined. Amplification products can be detected with a polynucleotideprobe that hybridizes, under conditions suitable for a polymerase chainreaction, with an Ehrlichia canis polynucleotide sequence. Theamplification product can be quantified by measuring a detection signalfrom the probe and comparing said detection signal to a second probedetection signal from a quantification standard. The quantificationstandard can be extracted in parallel with the test sample.

Methods of Treatment, Amelioration, or Prevention of a Disease Caused byE. canis

In one embodiment of the invention, a DIVA polypeptide, polynucleotideor antibody of the invention could be used to treat, ameliorate, orprevent a disease caused by E. canis. If, however, a DIVA polypeptide isused to treat, ameliorate, or prevent a disease caused by E. canis, itcould not, thereafter, be used as a DIVA polypeptide for the detectionand differentiation of infected, non-vaccinated, and vaccinated animalsbecause a vaccinated animal's immune system could recognize the DIVAantigen used for vaccination. However, a DIVA polypeptide that does notcross-react with antibodies to the DIVA polypeptide used for treatment,amelioration or prevention of a disease caused by E. canis may still beused as an E. canis DIVA antigen.

For example, if SEQ ID NO:2 or a fragment thereof is used as a vaccine,then SEQ ID NOs:4, 6, 8, 10, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or combinations thereof can beused as a DIVA polypeptide, if they do not cross-react with antibodiesspecific for SEQ ID NO:2. Presently, none of SEQ ID NOs:10, 22-33 areused in a commercial subunit E. canis vaccine and SEQ ID NOs:10, 22-33do not detect E. canis-specific antibodies in animals vaccinated withwhole inactivated E. canis cells. Therefore, DIVA polypeptide selectionis not presently an issue. However, those of skill in the art are awareof the composition of E. canis vaccines. If a commercial E. canissubunit vaccine were to comprise SEQ ID NOs:10, 22-33, then one of skillin the art would avoid use of SEQ ID NOs:10, 22-33 (if necessary due tothe generation of E. canis antibodies specific for SEQ ID NOs:10, 22-33)to differentiate vaccination status and would instead use other E. canisDIVA antigens.

Therefore, the DIVA polypeptides, polynucleotides, and antibodies couldbe used in two different ways: (1) as compositions for the prevention,treatment, or amelioration of a disease or infection caused by E. canis;and (2) as an E. canis DIVA antigen for the detection anddifferentiation of animals that are vaccinated; non-vaccinated; infectedor not infected with E. canis.

Polypeptides, polynucleotides, and antibodies of the invention can beused to treat, ameliorate, or prevent a disease caused by E. canis. Forexample, an antibody, such as a monoclonal antibody of the invention orfragments thereof, can be administered to an animal, such as a human. Inone embodiment of the invention an antibody or fragment thereof isadministered to an animal in a pharmaceutical composition comprising apharmaceutically acceptable carrier. A pharmaceutical compositioncomprises a therapeutically effective amount of an antibody or fragmentsthereof. A therapeutically effective amount is an amount effective inalleviating the symptoms of E. canis infection or in reducing the amountof E. canis organisms in a subject.

Polypeptides or polynucleotides of the invention can be present in animmunogenic composition and used to elicit an immune response in a host.An immunogenic composition is capable of inducing an immune response inan animal. An immunogenic polypeptide or polynucleotide composition ofthe invention is particularly useful in sensitizing an immune system ofan animal such that, as one result, an immune response is produced thatameliorates or prevents the effect of E. canis infection. Theelicitation of an immune response in animal model can be useful todetermine, for example, optimal doses or administration routes.Elicitation of an immune response can also be used to treat, prevent, orameliorate a disease or infection caused by E. canis. An immune responseincludes humoral immune responses or cell mediated immune responses, ora combination thereof. An immune response can also comprise thepromotion of a generalized host response, e.g., by promoting theproduction of defensins.

The generation of an antibody titer by an animal against E. canis can beimportant in protection from infection and clearance of infection.Detection and/or quantification of antibody titers after delivery of apolypeptide or polynucleotide can be used to identify epitopes that areparticularly effective at eliciting antibody titers. Epitopesresponsible for a strong antibody response to E. canis can be identifiedby eliciting antibodies directed against E. canis polypeptides ofdifferent lengths. Antibodies elicited by a particular polypeptideepitope can then be tested using, for example, an ELISA assay todetermine which polypeptides contain epitopes that are most effective atgenerating a strong response. Polypeptides or fusion proteins thatcontain these epitopes or polynucleotides encoding the epitopes can thenbe constructed and used to elicit a strong antibody response.

A polypeptide, polynucleotide, or antibody of the invention can beadministered to a mammal, such as a mouse, rabbit, guinea pig, macaque,baboon, chimpanzee, human, cow, sheep, pig, horse, dog, cat, or toanimals such as chickens or ducks, to elicit antibodies in vivo.Injection of a polynucleotide has the practical advantages of simplicityof construction and modification. Further, injection of a polynucleotideresults in the synthesis of a polypeptide in the host. Thus, thepolypeptide is presented to the host immune system with nativepost-translational modifications, structure, and conformation. Apolynucleotide can be delivered to a subject as “naked DNA.”

Administration of a polynucleotide, polypeptide, or antibody can be byany means known in the art, including intramuscular, intravenous,intrapulmonary, intramuscular, intradermal, intraperitoneal, orsubcutaneous injection, aerosol, intranasal, infusion pump, suppository,mucosal, topical, and oral, including injection using a biologicalballistic gun (“gene gun”). A polynucleotide, polypeptide, or antibodycan be accompanied by a protein carrier for oral administration. Acombination of administration methods can also be used to elicit animmune response. Antibodies can be administered at a daily dose of about0.5 mg to about 200 mg. In one embodiment of the invention antibodiesare administered at a daily dose of about 20 to about 100 mg.

Pharmaceutically acceptable carriers and diluents for therapeutic useare well known in the art and are described in, for example, Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro ed. (1985)).The carrier should not itself induce the production of antibodiesharmful to the host. Such carriers include, but are not limited to,large, slowly metabolized, macromolecules, such as proteins,polysaccharides such as latex functionalized SEPHAROSE®, agarose,cellulose, cellulose beads and the like, polylactic acids, polyglycolicacids, polymeric amino acids such as polyglutamic acid, polylysine, andthe like, amino acid copolymers, peptoids, lipitoids, and inactive,avirulent virus particles or bacterial cells. Liposomes, hydrogels,cyclodextrins, biodegradable nanocapsules, and bioadhesives can also beused as a carrier for a composition of the invention.

Pharmaceutically acceptable salts can also be used in compositions ofthe invention, for example, mineral salts such as hydrochlorides,hydrobromides, phosphates, or sulfates, as well as salts of organicacids such as acetates, proprionates, malonates, or benzoates.Especially useful protein substrates are serum albumins, keyhole limpethemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanustoxoid, and other proteins well known to those of skill in the art.Compositions of the invention can also contain liquids or excipients,such as water, saline, phosphate buffered saline, Ringer's solution,Hank's solution, glucose, glycerol, dextrose, malodextrin, ethanol, orthe like, singly or in combination, as well as substances such aswetting agents, emulsifying agents, tonicity adjusting agents,detergent, or pH buffering agents. Additional active agents, such asbacteriocidal agents can also be used.

If desired, co-stimulatory molecules, which improve immunogenpresentation to lymphocytes, such as B7-1 or B7-2, or cytokines such asMIP1α, GM-CSF, IL-2, and IL-12, can be included in a composition of theinvention. Optionally, adjuvants can also be included in a composition.Adjuvants are substances that can be used to nonspecifically augment aspecific immune response. Generally, an adjuvant and a polypeptide ofthe invention are mixed prior to presentation to the immune system, orpresented separately, but are presented into the same site of theanimal. Adjuvants can include, for example, oil adjuvants (e.g. Freund'scomplete and incomplete adjuvants) mineral salts (e.g. Alk(SO₄)₂;AlNa(SO₄)₂, AlNH₄(SO₄), Silica, Alum, Al(OH)₃, and Ca₃(PO₄)₂),polynucleotides (i.e. Polyic and Poly AU acids), and certain naturalsubstances (e.g. wax D from Mycobacterium tuberculosis, as well assubstances found in Corynebacterium parvum, Bordetella pertussis andmembers of the genus Brucella. Adjuvants which can be used include, butare not limited to MF59-0, aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637), referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/TWEEN®80 emulsion.

The compositions of the invention can be formulated into ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, injectable formulations, mouthwashes, dentrifices, andthe like. The percentage of one or more polypeptides, polynucleotides,or antibodies of the invention in such compositions and preparations canvary from 0.1% to 60% of the weight of the unit.

Administration of polypeptides, polynucleotides, or antibodies canelicit an immune response in the animal that lasts for at least 1 week,1 month, 3 months, 6 months, 1 year, or longer. Optionally, an immuneresponse can be maintained in an animal by providing one or more boosterinjections of the polypeptide, polynucleotide, or antibodies at 1 month,3 months, 6 months, 1 year, or more after the primary injection. Ifdesired, co-stimulatory molecules or adjuvants can also be providedbefore, after, or together with the compositions.

A composition of the invention comprising a polypeptide, polynucleotide,antibody, or a combination thereof is administered in a mannercompatible with the particular composition used and in an amount that iseffective to elicit an immune response as detected by, for example, anELISA. A polynucleotide can be injected intramuscularly to a mammal,such as a baboon, chimpanzee, dog, or human, at a dose of 1 ng/kg, 10ng/kg, 100 ng/kg, 1000 ng/kg, 0.001 mg/kg, 0.1 mg/kg, or 0.5 mg/kg. Apolypeptide or antibody can be injected intramuscularly to a mammal at adose of 0.01, 0.05, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 5 or 10 mg/kg.

Polypeptides, polynucleotides, or antibodies, or a combination thereofcan be administered either to an animal that is not infected with E.canis or can be administered to an E. canis-infected animal. Theparticular dosages of polynucleotide, polypeptides, or antibodies in acomposition will depend on many factors including, but not limited tothe species, age, gender, concurrent medication, general condition ofthe mammal to which the composition is administered, and the mode ofadministration of the composition. An effective amount of thecomposition of the invention can be readily determined using onlyroutine experimentation.

A method of monitoring an E. canis infection in a patient is alsoprovided. The method includes determining the level of anti-E. canisantibodies in a sample of a biological fluid from a patient sufferingfrom or at risk of an E. canis infection at a first time point usingpolypeptides of the invention. The level of anti-E. canis antibodies isdetermined in one or more samples of the biological fluid from thepatient at one or more different time points. The levels of anti-E.canis antibodies are determined at different time points such that theE. canis infection is monitored. The level or amount of anti-E. canisantibodies provide an indication of the success of treatment or therapy,or of progression of the infection.

All patents, patent applications, and other scientific or technicalwritings referred to anywhere herein are incorporated by reference intheir entirety. The invention illustratively described herein suitablycan be practiced in the absence of any element or elements, limitationor limitations that are not specifically disclosed herein. Thus, forexample, in each instance herein any of the terms “comprising”,“consisting essentially of”, and “consisting of” can be replaced witheither of the other two terms, while retaining their ordinary meanings.The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by embodiments,optional features, modification and variation of the concepts hereindisclosed may be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the description and the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

EXAMPLES Example 1 Preparation of Formalin Inactivated E. Canis forImmunization into Dogs

E. canis was grown in canine cell culture using methods described in theliterature. See e.g., Breitschwerdt, Antimicrobial Agents andChemotherapy, 1998, Vol 42:362-368. Using light microscopy, 030 cellswere estimated to be greater than 80% infected by E. canis. Two litersof E. canis infected cell culture were collected, centrifuged and thepellet retained yielding 7.31 gms of material (wet weight). It ispresumed water made up 80% of the weight of the material, giving anestimated dry weight of 1.462 gms (20% of the weight of the material).The cell pellet was resuspended to 20 mg/ml in PBS (dry weight) for atotal volume of 73 ml.

To this resuspended cell pellet, 0.73 ml of formalin solution was added(Sigma Catalog HT50-1-2 Formalin Solution 10%, neutral buffered) for afinal formaldehyde concentration of 0.04%. The solution was stirredovernight at 4° C. The inactivated mixture was centrifuged and the cellpellet retained. The pellet was washed by resuspension into 250 mls ofPBS. The material was collected by centrifugation and the wash wasrepeated one time.

The washed cell pellet was resuspended into 73 mls of PBS. The samplewas aliquoted to 73 screw cap vials and frozen at −80° C. Each vialcontains 20 mgs (dry weight) of formalin inactivated E. canis cellculture, suitable for combining with the appropriate adjuvant forimmunization into animals.

Example 2 Preparation of formalin inactivated E. canis with twodifferent adjuvants, protocol for the immunization of beagles with E.canis antigen, and testing of sera from immunized beagles using SNAP®3Dx® (reversible flow chromatographic assay)

The preparation of antigen with aluminum hydroxide adjuvant is atechnique well known to those skilled in the art. For example see“Antibodies, A Laboratory Manual”, Cold Spring Harbor Press, 1988, pp99.

For immunization into dogs (laboratory beagles), two sets of doses wereprepared with aluminum hydroxide adjuvant prepared as described aboveand two sets of doses were prepared with Ribi adjuvant (Corixa Corp.,Seattle Wash.) using the protocol described by the manufacturer. Eachdose contained approximately 20 mg of formalin inactivated E. canis cellculture (dry weight).

Kennel kept laboratory beagles were selected for immunization with theE. canis formalin inactivated antigen. Two groups of two dogs each; witheach group using a different adjuvant were dosed with the formalininactivated E. canis preparation (aluminum oxide or Ribi). On day 0 all4 dogs were found to be sero-negative using both the SNAP® 3Dx®(reversible flow chromatographic assay) diagnostic as well as westernblot analysis using E. canis organism.

The IACUC committee of Covance Research Products Inc. approved theprotocol for immunization of laboratory beagles. Dogs were vaccinated ondays 0, 28 and 56 with weekly 1 ml bleeds being monitored using SNAP®3Dx® (reversible flow chromatographic assay). All dogs were dosed withthe appropriate test article subcutaneously in the dorsoscapular area.All four animals seroconverted to a positive test on SNAP®3Dx®(reversible flow chromatographic assay) E. canis by day 42. Productionbleeds were taken on days 42 and 70 (approximately 50 ml blood thatyielded approximately 25 ml sera).

FIG. 1 shows SNAP®3Dx® Assay (reversible flow chromatographic assay)evaluation of laboratory beagles. The SNAP® device (reversible flowchromatographic assay) was used as described by manufacturer. “Pre”sample is from day 0. “Post” sample is from day 42. The E. canispositive spot becomes positive in all 4 dogs for the day 42 sample.Similar results were observed for the day 70 sample.

Experiments with a third vaccine comprising a third adjuvant, BCG,(Calbiochem of EMD Biosciences, Inc. San Diego, Calif.) revealed similarresults. Preparation of the third vaccine was identical to thepreparations described for the Ribi adjuvant vaccine described aboveexcept: 1) formalin inactivation was for 24 hrs at 4 C, and 2) 1 mg ofBCG was added. The vaccination schedule was day 0, day 14, with weeklybleeds assayed for reactivity with E. canis proteins.

Example 3 Enrichment of E. canis from Cell Culture Using PERCOLL®Gradients

For DNA isolation and western blot analysis, E. canis was enriched fromcell culture using PERCOLL® density gradients. The process of isolatingintracellular pathogens from cell culture, such as Ehrlichia, is atechnique well known to those skilled in the art. For example, see Akiraet al. (1982) Purification of Rickettsia tsutsugamushi by PERCOLL®density gradient centrifugation, Microbiol. Immunol., 26:321-328.

A typical E. canis enrichment began with 1.5 liters of infected cellculture (see above). The cells were centrifuged 6,000×g, the cell pelletretained and the supernatant discarded. The cell pellet was resuspendedinto 20 ml of PBS that was followed by a second centrifugation. Thesupernatant was discarded and supernatant retained. The pellet was thenresuspended into 20 ml of PBS, sonicated for 5 seconds at 20 kHz, powersetting 1.5 using a Branson sonicator. The sample was then centrifugedat 500×g for 5 minutes to pellet large debris.

PERCOLL® was added to the supernatant to a final concentration of 32%(4.5 ml of PERCOLL® with 10 ml of sample). The sample was loaded intoOak Ridge tubes compatible with a 70.1 Ti ultracentrifuge rotor, andcentrifuged for 30 minutes at 63,000×g. The opaque band was collectedusing a Pasteur pipette. The opaque band is highly enriched forEhrlichia (confirmed using light microscopy of the collected sample).After a 1:4 dilution with PBS, the sample was aliquoted and centrifugedat 12,000×g. The supernatant was discarded and the Ehrlichia pelletstored at −80° C.

Example 4 Testing of Sera or Plasma from Vaccinated and Infected Dogs byWestern Blot

The use of 1-dimensional SDS-PAGE gel analysis and 2-dimensional gelanalysis (1^(st) dimension isoelectric focusing, 2^(nd) dimensionSDS-PAGE) is well known to those skilled in the art. For example seeCurrent Protocols in Molecular Biology, eds. F. M. Ausubel et al., JohnWiley & Sons Inc., 1997, pages 10.2.2-10.3.11. The use of western blotsto analyze proteins separated using these methods are well known tothose skilled in the art. For example see Current Protocols in MolecularBiology, eds. F. M. Ausubel et al., John Wiley & Sons Inc., 1997, pages10.8.1-10.8.116.

Initial work was performed using western analysis of proteins separatedwith 1D gels (data not shown), followed by western analysis of proteinsseparated using 2D gels. Proteins from whole E. canis harvested fromcell culture were analyzed using 2D gel electrophoresis (materials andreagents used as described by the manufacturer; Bio-Rad Life SciencesResearch, Hercules, Calif. 94547). The amount of sample to load per gelwas determined empirically (see FIG. 2). The proteins were blotted tonitrocellulose and probed using canine sera from laboratory beagles atday 0, dogs vaccinated with formalin inactivated E. canis antigen (seeabove), or sera from animals infected with E. canis (see FIGS. 3, 4 &5).

Positive canine sera and plasma was isolated from dogs infected with E.canis. E. canis infection was verified by western analysis oflymphocytes harvested from whole blood from these dogs, and confirmed byuse of the IDEXX SNAP®3Dx® assay (reversible flow chromatographic assay)with canine sera or plasma (commercially available from IDEXXLaboratories Inc., used as described by the manufacturer).

For western blot analysis proteins were separated using 1D SDS-PAGE or2D isoelectric focusing/SDS-PAGE gels followed by electo-blotting of theproteins from the gels to nitrocellulose. The nitrocellulose blots wereincubated in a blocking solution of 2.5% non fat dry milk dissolved intoTris buffered saline (pH 7.5), 0.05% TWEEN® 20 (polysorbate). Caninesera or plasma was diluted to the titer as described into buffercontaining an E. coli lysate to block non-specific binding with 30%normal calf sera and incubated for 2 hrs at room temperature or overnight at 4° C. After washing 3 times in TBS-TWEEN® (polysorbate)(0.05%), the blots were transferred to a buffer containing 50% fetalcalf sera, 50% TBS-TWEEN® (polysorbate)-Kathon (0.05% & 0.5%respectively) to prevent nonspecific binding of a rabbit anti-canine Fcpolyclonal antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA 19390). The rabbit anti-canine Fc polyclonalantibody conjugate was diluted 1:5,000. The gels were washed 3 timeswith TBS TWEEN® (polysorbate) (0.05%), one time with TBS, and thepresence of HRP detected using ECL western Blotting Detection Reagents(Amersham Biosciences, Piscataway, NJ 08855-1327) used as described bymanufacturer. Digital images of exposed X-ray film were captured using aGelDoc 2000 (Bio-Rad Inc.).

Example 5 Isolation of DNA from E. Canis and Construction of a LambdaExpression Library and Screening of the E. Canis Lambda ExpressionLibrary for Clones Having DIVA Activity

The preparation and screening of lambda expression libraries is atechnique well known to those skilled in the art. For example, seeCurrent Protocols in Molecular Biology, eds. F. M. Ausubel et al., JohnWiley & Sons Inc., 1997, pages 5.1 through 5.8.6. For the constructionof the expression library, genomic DNA was purified from E. canisisolated from cell culture by PERCOLL® gradient centrifugation (seeabove). DNA was purified using a genomic DNA purification kit fromQiagen Sciences (Germantown, Md.). A Lambda ZAP® II predigestedEcoRI/CIAP Vector Kit (Stratagene Corp., La Jolla, Calif. 92037) wasused as specified by the manufacturer for construction of the library.E. canis genomic DNA was partially digested with TSP509 and fragmentsranging from 2-6 kb were isolated using agarose gel electrophoresis andligated into the lambda vector. Phage were packaged and grown asspecified by the manufacturer.

Approximately 120,000 individual lambda plaques were screened forbinding to sera isolated from dogs identified as positive for infectionwith E. canis, but negative for reactivity with sera from animalsvaccinated with formalin inactivated E. canis (see above). From theinitial screen 84 individual plaques were identified as having thisactivity.

Lambda plaques were subjected to two rounds of plaque purification andretested to verify positive reactivity with sera from E. canis infectedanimals, negative reactivity when screened with sera from vaccinatedanimals.

Isolated lambda plaques were screened for cross reactivity with serafrom animals identified as being seropositive for Anaplasmaphagocytophilia, Borrelia burgdorferi (causative agent of Lyme disease),Rickettsia rickettsii (causative agent of Rocky Mountain Spotted Fever),Leptospira interrogans and Dirofilaria immitis (causative agent ofcanine heartworm).

At the end of the screening process, 43 lambda plaques were found toreact with sera from animals infected with E. canis that did not reactwith sera from vaccinated dogs or sera from dogs infected with othercanine pathogens (see above).

Using the ZAP® feature of the cloning vector as per the manufacturersinstructions, inserts into the lambda vector were converted to plasmids.The plasmids were transformed into the E. coli strain XL-1 blue forprotein expression and analysis of encoded proteins by western blot. Theends of the E. canis DNA inserts were subjected to DNA sequence analysisusing T7 and T3 sequencing primers.

Sequence information from both the T7 and T3 reactions for all 43 cloneswas submitted for BLAST analysis to the NCBI website. Results weretabulated in an excel format. Based on sequence identity between theclone and the available shotgun genome sequence for E. canis (NCBI:NZ_AAEJ01000001), segments of genomic DNA for each clone wereidentified. Individual clones sharing common genes were grouped forfurther analysis by western blot using pools of infected and vaccinatedcanine sera. Based on similar banding patterns, duplicate clones wereeliminated. Any clones showing reactivity to both sets of sera wereeliminated. As a result of this analysis, 23 clones were selected forfurther evaluation. The grouping of the clones and the common antigenper group is shown in Table 2.

TABLE 2 Common Antigen Clone Number(s) 120 kDa Antigen 2, 10, 17, 33,35, 79 Heat Shock Proteins 4, 9, 24, 66 ATPase 7, 84 Ribosomal ProteinL1 21, 47, 65 200 kDa Antigen 26, 55, 76 Hypothetical Protein 75Pyruvate Dehydrogenase  5 Ribosomal Protein (50S)  6 Unknown 57Transcriptional Regulator 82

Example 6 Western Blot Analysis Using Individual E. Canis PositiveCanine Serum Samples

All 23 clones were analyzed on individual SDS-PAGE gels. Each gel wastransferred to nitrocellulose and subjected to western blotting usingindividual samples of canine sera from dogs that were only positive forE. canis infections by ELISA/SNAP® (reversible flow chromatographicassay) testing. Canine serum was diluted 1:500 in the same diluentdescribed in Example 4 containing E. coli lysate and reactivity wasdetected using standard colorimetric horseradish peroxidase techniques(Opti-4CN, Bio-Rad). A total of thirteen individual canine serum sampleswere evaluated. Blots were compared across samples to determine thenumber of dogs showing reactivity to a predominant band or set of bandsper clone. The results are summarized in Table 3 and FIG. 6 (cloneslisted in bold are depicted in the figure).

TABLE 3 Common Antigen Clone Number(s) Positive Reactors 120 kDa Antigen2, 10, 17, 33, 35 13/13 Heat Shock Proteins 9 12/13 ATPase 7, 84 12/13Ribosomal Protein L1 21, 47, 65 12/13 200 kDa Antigen 26, 55, 76 12/13

All 23 clones were also analyzed by western blot using pooled caninesera that had tested positive for other vector-borne infectiousdiseases. Samples testing positive by ELISA or SNAP® (reversible flowchromatographic assay) for the following single infections wereevaluated: Heartworm, Lyme, Anaplasma phagocytophilum, or E. ewingii.None of the clones identified in the table above showed cross-reactivitywith positive canine sera for these other vector-borne infections.

Example 7 Identification of Relevant Gene Segments Encoding E. CanisDIVA Antigens

a. 120 kDa Antigen

This antigen was previously described by Yu et al. (J Clin Microbiol.2000 January; 38(1):369-74; see also, McBride et al., 2000 Infec. Immun.68:13) and shown to be useful in the diagnosis of E. canis infections indogs. This antigen has been described as both “p120” and “p140” E. canisantigen. See, id. Yu et al. explains that a recombinant proteinexpressed by the p120 gene has a molecular size of 140 kDa on a sodiumdodecyl sulfate gel, which is larger than the predicted molecular massof the protein. See, Yu et al., page 373. The Walker group (Yu et al.,and McBride et al.) refer to the protein both as E. canis p120 and p140.Therefore, this disclosure uses both p120 and p140 interchangeably todescribe this protein. The accession number for the E. canis p120/140gene is AF112369 and the associated protein is AAD34330. See also,accession no. YP302666. Clones 2, 10, 17, and 33 contain full-lengthsegments of the 120 kDa antigen gene. Clone 35 may contain a truncationof this gene. (See, SEQ ID NOs:1 and 2).

This gene was amplified from E. canis genomic DNA and subcloned into apET expression system with a 6-His tag according to the manufacturer'sinstructions (Invitrogen). Sequencing results of this plasmid exactlymatched the gene sequence encoding the protein shown in SEQ NO:ID 2,from amino acids 58 to 589. Protein lysates from BL21 bacteria inducedto express this protein were analyzed by western blotting with infectedcanine sera and compared to western blots probed with sera from animalsvaccinated with formalin inactivated E. canis cells. Consistent withprevious findings, only sera from infected dogs recognized this proteinof the expected molecular weight (data not shown).

P120 has a 36 amino acid motif that is repeated 14 times. See, SEQ IDNO:15. The repeated portion (underlined region in SEQ ID NO:15 is a 60kD peptide). SEQ ID NO:16 shows the aligned 14 repeats. SEQ ID NO:17shows the consensus sequence of the 14 repeats.

One embodiment of the invention provides a polypeptide comprising:

(SEQ ID NO: 17) KEEX₁TPEVX₂AEDLQPAVDX₃SX₄EHSSSEVGX₅KVSX₆TS.

-   -   Where        -   X₁=S or N        -   X₂=K or R        -   X₃=G, D, or S        -   X₄=V or I        -   X₅=E or K        -   X₆=E or K            Another embodiment of the invention provides a multimeric            polypeptide where SEQ ID NO:17 is repeated two or more            times. The multimeric polypeptide can also comprise one or            more heterologous polypeptides.

In another embodiment, the invention provides a polypeptide of SEQ IDNO:21, XPEVKAEDLQPAVDGSVEHX, wherein each of the X's=0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

b. 200 kDa Antigen

This antigen was previously described by McBride et al. (J ClinMicrobiol. 2001 January; 39(1):315-22) and shown to be useful in thediagnosis of ehrlichiosis. The accession number for this gene isAF252298 and associated protein AAK01145. A portion of this proteinsequence is associated with a published patent (SEQ ID NO:2 of U.S. Pat.No. 6,355,777, accession number AAE96254). We have identified adifferent region of this protein that serves as diagnostic antigen forehrlichiosis and a DIVA reagent. The portion of the gene spans fromnucleotide 1081 of AF252298 through to the end, nucleotide 4266. (SeeSEQ ID NOs:3 and 4).

This gene was amplified from E. canis genomic DNA and subcloned into apET expression system with a 6-His tag according to the manufacturer'sinstructions (Invitrogen). Sequencing results of this plasmid exactlymatched the gene sequence encoding the protein shown in SEQ ID NO:4,from amino acids 1 to 1061. Protein lysates from BL21 bacteria inducedto express this protein were analyzed by western blotting with infectedcanine sera and compared to western blots probed with sera from animalsvaccinated with formalin inactivated E. canis. Consistent with previousfindings, only sera from infected dogs recognized this protein of theexpected molecular weight (data not shown).

c. ATPase

This gene (Locus tag “Ecan02000699”) has been predicted by automatedcomputational analysis of the shotgun genome sequence of E. canis. Itcodes for a protein of more than 4000 amino acids (ZP_(—)00210575). TheE. canis DIVA screen identified two separate regions of this gene andits associated protein as potential immunodominant antigens and DIVAreagents. The segments of the protein identified in clones 84 and 7 areamino acids 1984-2774 and 2980-3740, respectively, of accession number46308382. (See SEQ ID NOs: 5, 6, 7, 8).

Both fragments of this gene was amplified from E. canis genomic DNA andsubcloned separately into a pET expression system with a 6-His tagaccording to the manufacturer's instructions (Invitrogen). Sequencingresults of this plasmid exactly matched the gene sequences associatedwith the proteins shown SEQ ID NOs:6 and 8, from amino acids 1 to 782and 1 to 746 respectively. Protein lysates from BL21 bacteria induced toexpress these proteins were analyzed by western blotting with infectedcanine sera and compared to western blots probed with sera from animalsvaccinated with formalin inactivated E. canis. Consistent with previousfindings, only sera from infected dogs recognized these proteins of theexpected molecular weight (data not shown).

d. Heat Shock Proteins

Although this clone contained a gene for the heat shock protein, GrpE,the gene sequence coding for the immunodominant antigen arises from ahypothetical protein sequence predicted by the automated computationalanalysis of the genome. Based on the molecular weight and pI of theprotein, the gene of interest in clone 9 is locus number “Ecan02000495”and the associated protein 46308954.

Because this protein is only predicted from the computer annotation ofthe genome and has not been previously identified from E. canisorganisms as an immunodominant protein, this is the first evidence thatthis gene is expressed in E. canis and stimulates an immune response inthe infected canine host. The protein will be identified as the p16antigen (see SEQ ID NO: 9 and 10).

This gene was amplified from the pBlueScript vector containing thegenomic DNA of interest and subcloned into a pET expression system witha 6-His tag according to the manufacturer's instructions (Invitrogen).Sequencing results of this plasmid exactly matched the gene sequenceassociated with locus number “Ecan02000495”. Protein lysates from BL21bacteria induced to express this protein were analyzed by westernblotting with infected canine sera and compared to western blots probedwith sera from animals vaccinated with formalin inactivated E. canis.Consistent with previous findings, only sera from infected dogsrecognized this protein of the expected molecular weight (see FIG. 7).

e. Ribosomal Protein L1

This gene is identified by the locus tag “Ecan02000476” from the E.canis genome. The associated protein has the accession number ZP00211130 (see SEQ ID NOs:11 and 12). The identification of this proteinhas been predicted based on automated computational analysis of thegenome. A BLAST analysis of this protein reveals that the sequence isabout 70% identical to a surface protein of E. chaffeensis (Accessionnumber 4894576). Immunoreactivity to the E. chaffeensis protein haspreviously been reported by Yu et al., (J Clin Microbiol. 1999 August;37(8):2568-75). The E. chaffeensis protein (Accession number 4894576) isreferred to as the 106 kDa protein precursor.

f. Possible Non-120 kDa Antigens

Within the genomic fragment containing the gene for the 120 kDa antigen,other genes are present that may also be immunodominant and DIVAreagents. For instance, clone 10 produces a different banding pattern onwestern blots probed with infected sera, compared to clones containingthe 120 kDa antigen alone. Clone 10 contains genetic information for theVirD4 components of a Type IV secretory pathway and this gene sequenceis identified by the locus tag “Ecan02000624”. This gene codes for aprotein of 723 amino acids (ZP_(—)00211244), but only a portion of thisprotein appears to be expressed by clone 10, as determined by themolecular weight of the protein identified on the gel (see SEQ ID NOs:13and 14).

Example 8 Evaluation of E. canis P140 Peptides

Sera from beagles immunized with formalin inactivated E. canis (vaccinesamples) were tested using a microtiter-plate based immunoassay preparedusing synthetic peptides derived from E. canis p140 protein (also knownas p120, see Example 7).

Preparation of Formalin Inactivated E. Canis and Immunization of Beagleswere Described in Examples 1 and 2. Samples from immunized beagles weretested using microtiter-plate based immunoassays prepared usingsynthetic peptides (SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20) inindirect and direct assay formats.

Indirect Assay Format

Samples were tested using microtiter-plate based immunoassays preparedusing the synthetic peptides (SEQ ID NO:18, SEQ ID NO:19 and SEQ IDNO:20). Individual peptides were immobilized on microtiter wells bydirect adsorption. A dilution of the test sample (1:100) was added tothe microtiter well and unbound antibody was removed by washing.Antibody bound to the immobilized peptide was detected by reaction withan anti-species, in this case canine, horseradish peroxidase (HRPO)conjugate (1:2000 dilution), washing and addition of a HRPO substrate.The absorbance (A650) of individual microtiter wells was determinedusing a microtiter plate reader.

Direct Assay Format

Individual peptides (SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20) wereconjugated to bovine serum albumin and immobilized on microtiter wellsby direct adsorption. Synthetic peptides (SEQ ID NO:18, SEQ ID NO:19 andSEQ ID NO:20) were conjugated to an indicator reagent, horseradishperoxidase (HRPO). The test sample and the immunoassay peptide/indicatorwere added to a microtiter well coated with the corresponding peptide,which was incubated and washed. Antibody bound to the immobilizedpeptide and the peptide/indicator reagent was detected by addition of anHRPO substrate reagent. The absorbance (A650) of individual microtiterwells was determined using a microtiter plate reader.

Assay results are shown in Table 4. The positive control (PC, ID1049:16E) and negative control (NC, 3818:57B) were known E. canispositive and negative serum samples, respectively. All samples weretested using the commercially available SNAP® 4Dx® test (reversible flowchromatographic assay) for E. canis antibody. Results for sequentialtemporal samples from 6 dogs (CVYDEH, CWMBDC, CVXCSM, CWMAXK, CVSCVA andCVXCAP) receiving the formalin inactivated E. canis antigen formulatedusing different adjuvants are shown for day 0 to day 42post-immunization. Results of the SNAP® 4Dx® test demonstrate that anantibody response was induced in the vaccinated animals. None of theserum samples from vaccinated animals was reactive in the direct assayformat. Several samples (for example from dog CWMAXK) had highbackground reactions in the indirect assay format.

The results demonstrate that antibody induced as a result ofimmunization using formalin inactivated vaccine was significantlynon-reactive to the synthetic peptides derived from an E. canis p140protein. (SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20).

TABLE 4 Reaction of sera from dogs immunized with formalin inactivatedE. canis antigen measured using microtiter assays prepared usingpeptides derived from E. canis p140 protein. (SEQ ID NO: 18, SEQ ID NO:19 and SEQ ID NO: 20). Indirect Plate Results Direct Plate Results(A650) (A650) 4Dx ® E. canis SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDSample Result NO: 18 NO: 19 NO: 20 NO: 18 NO: 19 NO: 20 1049:16E (PC)0.72 2.071 2.075 1.867 2.049 1.821 1.495 3818:57B (NC) N 0.051 0.0580.050 0.034 0.033 0.035 CVYDEH day 0 N 0.050 0.062 0.045 0.034 0.0340.035 day 7 N 0.048 0.052 0.042 0.033 0.032 0.036 day 14 N 0.051 0.0550.048 0.036 0.034 0.038 day 21 N 0.044 0.062 0.051 0.035 0.034 0.040 day28 0.04 (vw+) 0.054 0.073 0.055 0.036 0.033 0.034 day 35 0.07 (vw+)0.049 0.058 0.047 0.033 0.035 0.039 day 42 N 0.051 0.059 0.053 0.0340.035 0.040 CWMBDC day 0 0.08 0.054 0.085 0.082 0.035 0.033 0.038 day 70.20 0.064 0.078 0.072 0.038 0.035 0.035 day 14 0.30 0.058 0.081 0.0850.038 0.033 0.040 day 21 0.24 0.051 0.101 0.078 0.037 0.040 0.039 day 280.22 0.049 0.082 0.073 0.034 0.036 0.033 day 35 0.17 0.043 0.068 0.0810.033 0.040 0.035 day 42 0.11 0.044 0.071 0.074 0.031 0.034 0.031 CVXCSMday 0 N 0.049 0.082 0.051 0.033 0.035 0.034 day 7 N 0.038 0.076 0.0520.034 0.033 0.037 day 14 N 0.044 0.069 0.049 0.033 0.032 0.038 day 210.10 (w+) 0.038 0.054 0.045 0.035 0.035 0.036 day 28 0.10 (w+) 0.0440.060 0.049 0.036 0.033 0.035 day 35 0.08 (vw+) 0.040 0.062 0.053 0.0340.035 0.041 day 42 0.05 (vw+) 0.041 0.057 0.049 0.033 0.035 0.036 CWMAXKday 0 0.07 (vw+) 0.043 0.078 0.054 0.034 0.039 0.037 day 7 0.41 0.0820.475 0.413 0.034 0.034 0.045 day 14 0.44 0.049 0.782 0.607 0.034 0.0350.044 day 21 0.36 0.092 0.587 0.440 0.033 0.037 0.038 day 28 0.39 0.0630.407 0.258 0.037 0.034 0.038 day 35 0.41 0.056 0.286 0.212 0.036 0.0340.037 day 42 0.35 0.048 0.196 0.155 0.034 0.034 0.041 CVSCVA day 0 0.10(w+) 0.039 0.084 0.084 0.033 0.033 0.038 day 7 0.37 0.040 0.107 0.0660.032 0.032 0.036 day 14 0.14 0.053 0.151 0.062 0.035 0.033 0.039 day 210.33 0.057 0.131 0.072 0.035 0.033 0.034 day 28 0.29 0.049 0.104 0.0580.035 0.034 0.036 day 35 0.36 0.043 0.108 0.079 0.034 0.039 0.040 day 420.32 0.047 0.117 0.044 0.033 0.036 0.037 CVXCAP day 0 N 0.041 0.0650.040 0.032 0.035 0.032 day 7 0.34 0.058 0.106 0.068 0.036 0.033 0.033day 14 0.30 0.087 0.150 0.112 0.034 0.035 0.039 day 21 0.35 0.065 0.1200.086 0.039 0.036 0.041 day 28 0.19 0.054 0.103 0.059 0.035 0.036 0.032day 35 0.18 0.046 0.092 0.047 0.033 0.033 0.039 day 42 0.19 0.051 0.0670.047 0.035 0.035 0.038

Example 9

Sera from known E. canis positive and negative dogs was tested using amicrotiter-plate based immunoassay prepared using the synthetic peptidesobtained from E. canis protein p140 protein (also known as p120, seeExample 7)

E. canis positive and negative field samples were obtained and testedusing the SNAP® 4Dx® test (reversible flow chromatographic assay) forantibody to E. canis. Samples were then tested using indirect and directmicrotiter plate format assays produced using synthetic peptides derivedfrom the E. canis P140 protein (SEQ ID NO:18, SEQ ID NO:19 and SEQ IDNO:20).

Indirect Assay Format

Samples were tested using microtiter-plate based immunoassays preparedusing the synthetic peptides (SEQ ID NO:18, SEQ ID NO:19 and SEQ IDNO:20). Individual peptides were immobilized on microtiter wells bydirect adsorption. A dilution of the test sample (1:100) was added tothe microtiter well and unbound antibody was removed by washing.Antibody bound to the immobilized peptide was detected by reaction withan anti-species, in this case canine, horseradish peroxidase (HRPO)conjugate (1:2000 dilution), washing and addition of a HRPO substrate.The absorbance (A650) of individual microtiter wells was determinedusing a microtiter plate reader.

Direct Assay Format

Individual peptides (SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20) wereconjugated to bovine serum albumin and immobilized on microtiter wellsby direct adsorption. The synthetic peptides (SEQ ID NO:18, SEQ ID NO:19and SEQ ID NO:20) were conjugated to the indicator reagent, horseradishperoxidase (HRPO). The test sample and the immunoassay peptide/indicatorwere added to a microtiter well coated with the corresponding peptide,which was incubated and washed. Antibody bound to the immobilizedpeptide and the peptide/indicator reagent was detected by addition of anHRPO substrate reagent. The absorbance (A650) of individual microtiterwells was determined using a microtiter plate reader.

Table 4 shows results for E. canis positive and negative field samplestested using the indirect assay format. The positive control (PC, ID1049:16E) and negative control (NC, 3818:57B) were known E. canispositive and negative serum samples, respectively. Samples weredetermined to be E. canis antibody positive or negative using the SNAP®4Dx® test (reversible flow chromatographic assay). Assay results areshown for microtiter plate format assays made using peptide reagents(SEQ ID:18, SEQ ID:19 and SEQ ID:20).

Table 5 shows results for E. canis positive and negative field samplestested using the direct assay format. The positive control (PC, ID1049:16E) and negative control (NC, 3818:57B) were known E. canispositive and negative serum samples, respectively. Samples weredetermined to be E. canis antibody positive or negative using the SNAP®4Dx® test (reversible flow chromatographic assay). Assay results areshown for microtiter plate format assays made using peptide reagents(SEQ ID:18, SEQ ID:19 and SEQ ID:20).

TABLE 5 E. canis positive and negative field samples tested using theindirect microtiter plate format assay constructed using P140 peptides(SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20). Absorbance at 650 nM4Dx ® SEQ ID SEG ID SEQ ID Sample Result No: 18 NO: 19 no: 20 1049:16E(PC) 2.292 2.735 2.584 3818:57B (NC) 0.051 0.065 0.045 EC+ HP 127 0.070.042 0.050 0.038 EC+ HP 143 0.08 2.867 2.825 2.731 EC+ HP 147 0.092.370 2.661 2.658 EC+ HP 151 0.21 2.176 2.093 2.535 EC+ HP 161 0.181.708 2.178 2.551 EC+ HP 165 0.08 2.690 2.492 2.525 EC+ HP 172 0.070.229 0.902 2.197 EC+ HP 185 0.38 2.497 2.622 2.704 EC+ HP 186 0.262.899 2.979 2.794 EC+ HP 188 0.40 2.482 2.578 2.898 EC+ HP 190 0.212.484 2.534 2.632 EC+ HP 192 0.18 1.473 2.132 2.526 EC+ HP 194 0.432.583 2.429 2.539 EC+ HP 197 0.22 2.150 2.239 2.537 EC+ HP 201 0.362.449 2.472 2.519 EC+ HP 206 0.10 2.477 2.247 2.549 EC+ HP 207 0.082.030 2.359 2.369 EC+ HP 209 0.20 0.262 0.218 1.102 EC+ HP 213 0.211.471 1.662 2.406 EC+ HP 215 0.19 2.144 2.431 2.721 EC− HP 116 0.020.110 0.065 0.070 EC− HP 119 0.02 0.102 0.091 0.079 EC− HP 120 0.010.058 0.063 0.045 EC− HP 121 0.02 0.054 0.064 0.057 EC− HP 122 0.030.053 0.059 0.040 EC− HP 124 0.02 0.055 0.061 0.052 EC− HP 128 0.020.068 0.072 0.054 EC− HP 129 0.02 0.056 0.057 0.044 EC− HP 130 0.010.049 0.048 0.039 EC− HP 131 0.01 0.051 0.053 0.043 EC− HP 132 0.030.057 0.061 0.038 EC− HP 134 0.02 0.059 0.084 0.114 EC− HP 137 0.030.043 0.046 0.037 EC− HP 138 0.01 0.055 0.063 0.048 EC− HP 139 0.010.064 0.062 0.056 EC− HP 140 0.00 1.574 2.444 2.491 EC− HP 142 0.020.065 0.068 0.069 EC− HP 144 0.02 0.080 0.079 0.081 EC− HP 145 0.011.564 1.934 2.095 EC− HP 148 0.01 0.037 0.043 0.043

TABLE 6 E. canis positive and negative field samples tested using thedirect microtiter plate format assay constructed using P140 peptides(SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20). 3Dx ® Absorbance at650 nM SNAP SEQ ID SEQ ID SEQ ID Sample S-Bkg NO: 18 NO: 19 NO: 201049:16E (PC) 0.72 2.753 2.079 2.018 3818:57B (NC) Neg 0.034 0.035 0.0361049:16A E. canis pos 0.28 0.201 0.173 1.448 1049:16G E. canis pos 0.500.034 0.034 0.039 1049:16Q E. canis pos 0.39 2.308 1.933 2.151 1049:16UE. canis pos 0.56 0.627 2.038 2.254 1061:03B E. canis pos 0.49 0.0830.338 0.889 1061:03I E. canis pos 0.27 2.766 2.593 1.646 1177:21D E.canis pos 0.15 0.042 0.046 0.126 1177:21G E. canis pos 0.41 1.087 1.6751.835 1177:21K E. canis pos 0.34 0.681 1.930 2.010 1177:63O E. canis pos0.41 0.146 0.112 1.587 1183:85A E. canis pos 0.49 2.768 2.757 2.4761256:31I E. canis pos 0.23 0.044 0.086 0.143  813:91F E. canis pos 0.411.239 1.570 1.993  813:91I E. canis pos 0.41 0.212 0.517 1.646 EC 10 E.canis pos 0.37 0.236 0.302 0.465

The results demonstrate that antibody induced as a result of naturalinfection was reactive to the synthetic peptides derived from the E.canis p140 protein. (SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20).

Example 10 Testing of Sera from Known E. Canis Positive and NegativeDogs Using a Microtiter-Plate Based Immunoassay Prepared Using theSynthetic Peptides (SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24)Obtained from the E. Canis Protein p16 Protein. Assays were PerformedUsing the Indirect Assay Format Making Use of Anti-Canine HRPO Conjugateas the Indicator

Sera from six E. canis-antibody positive and three E. canis-antibodynegative canines were obtained from Sinclair Research (Columbia, MO).Serum samples were found to be positive or negative by testing using thelicensed reversible flow chromatographic binding assay IDEXX SNAP® 4Dx®test (reversible flow chromatographic assay) for E. canis antibody.Reversible flow chromatographic SNAP® assay (reversible flowchromatographic assay) results are shown in Table 7.

Samples were tested using microtiter-plate based immunoassays preparedusing synthetic peptides (SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24)derived from the E. canis p16 surface protein. The synthetic peptide wasimmobilized in Immulon microtiter wells at 0.25 ug/ml (SEQ ID NOs: 22and 23, or at 0.5 ug/ml (SEQ ID NO: 24)). A dilution of the test sample(1:100) was added to the microtiter well and unbound antibody wasremoved by washing. Antibody bound to the immobilized peptide wasdetected by reaction with an anti-species, in this case canine,horseradish peroxidase (HRPO) conjugate (1:2000 dilution), washing andaddition of HRPO substrate. The absorbance at 650 nm (A650) of fluid inindividual microtiter wells was determined using a microtiter-platereader.

Results:

Results for positive and negative samples are shown in Table 7. Positivesamples HP-319, HP-322, HP-326, HP-342, HP-354, HP-358 were reactive tothe peptide sequences shown in SEQ ID NO:22, SEQ ID NO:23 and SEQ IDNO:24. Negative samples HP-302, HP-303 and HP-306 were nonreactive tothe peptide sequences shown in SEQ ID NO:22, SEQ ID NO:23 and SEQ IDNO:24.

Conclusions:

The results demonstrate that antibody induced as a result of naturalinfection was reactive to the synthetic peptides derived from the E.canis p16 protein (SEQ ID NO:22, SEQ ID NO:23 and SEQ NO:24) in theindirect assay format described above.

TABLE 7 Assay results for positive and negative canine samples using E.canis synthetic peptide (SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24)coated microtiter wells and anti-species conjugate as indicator. A650 isabsorbance at 650 nm. The “4Dx ® SNAP ®” (reversible flowchromatographic assay) column presents the results from the reversibleflow chromatographic SNAP ® 4Dx ® assay (reversible flow chromatographicassay). Plate Results (A650) SEQ ID SEQ ID SEQ ID 4Dx NO: 22 NO: 23 NO:24 Sample SNAP A650 Result A650 Result A650 Result 1049:16E pos 1.733pos 2.309 pos 1.943 pos (PC) 3818:57B neg 0.046 neg 0.044 neg 0.041 neg(NC) HP-319 pos 1.274 pos 1.765 pos 0.755 pos HP-322 pos 1.247 pos 1.996pos 0.692 pos HP-326 pos 1.656 pos 2.159 pos 0.991 pos HP-342 pos 0.704pos 1.480 pos 0.277 pos HP-354 pos 1.220 pos 1.745 pos 0.573 pos HP-358pos 1.890 pos 2.270 pos 0.342 pos HP-302 neg 0.043 neg 0.043 neg 0.032neg HP-303 neg 0.036 neg 0.039 neg 0.029 neg HP-306 neg 0.044 neg 0.039neg 0.039 neg

Example 11 Testing of Sera from Known E. Canis Positive and NegativeDogs Using a Microtiter-Plate Based Immunoassay Prepared Using theSynthetic Peptides (SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24) Derivedfrom the E. Canis p16 Protein Sequence. Assays were Performed Using theDirect Assay Format Making Use of HRPO-Labeled Peptide as the Indicator

Sera from seven E. canis-antibody positive and three E. canis-antibodynegative canines were obtained from field dogs. Serum samples were foundto be positive or negative by testing using the licensed reversible flowchromatographic IDEXX SNAP® 3Dx® (reversible flow chromatographic assay)for E. canis antibody. Assay results are shown in Table 8.

Samples were tested using a microtiter-plate based immunoassay preparedusing the synthetic peptides (SEQ ID NO:22, SEQ ID NO:23 and SEQ N0:24)derived from the E. canis P16 surface protein. The synthetic peptideswere immobilized on microtiter plate wells at 1 ug/ml. Separatequantities of the synthetic peptides were conjugated to the indicatorreagent horseradish peroxidase (HRPO). The test sample and thepeptide:HRPO conjugate (1 ug/ml) were added to the peptide-coatedmicrotiter well, which was incubated and washed. Sample antibody boundto the immobilized peptide and the peptide::HRPO conjugate wasimmobilized in the microtiter well. This complex was detected byaddition of an HRPO substrate reagent. The optical density of individualmicrotiter wells was determined using a microtiter plate reader.

Results:

Results for positive and negative samples are shown in Table 8. Positivesamples 813:91I, 1049:16 A, 1049:16 U, 1061:03I, 1177:21 G, 1177:21 Kand 1177:63O were reactive to the peptide sequences shown in SEQ IDNO:22, SEQ ID NO:23 and SEQ ID NO:24. Negative samples 3818:57 A,3818:57 C and 3818:57 D were nonreactive to the peptide sequences shownin SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.

Conclusions:

The results demonstrate that antibody induced as a result of naturalinfection was reactive to the synthetic peptides derived from the E.canis p16 protein (SEQ ID NO:22, SEQ ID NO:23 and SEQ N0:24) in thedirect assay format described above.

TABLE 8 Assay results for positive and negative canine field samplesusing E. canis synthetic peptide-coated microtiter wells (SEQ ID NO: 22,SEQ ID NO: 23 and SEQ ID NO: 24) and E. canis syntheticpeptide-conjugates (SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24) asindicators. A650 is absorbance at 650 nm. The “3Dx ® SNAP ®” (reversibleflow chromatographic assay) column presents the results from thereversible flow chromatographic SNAP ® 3Dx ® assay (reversible flowchromatographic assay). Plate Results (A650) SEQ ID SEQ ID SEQ ID 3DxNO: 22 NO: 23 NO: 24 Sample SNAP A650 Result A650 Result A650 Result1049:16E pos 2.497 pos 2.908 pos 1.367 pos (PC) 3818:57B neg 0.038 neg0.041 neg 0.056 neg (NC) 813:91I pos 1.244 pos 1.828 pos 0.651 pos1049:16A pos 0.547 pos 0.819 pos 0.116 pos 1049:16U pos 2.653 pos 3.531pos 1.031 pos 1061:03I pos 0.665 pos 1.801 pos 0.127 pos 1177:21G pos1.484 pos 2.353 pos 0.444 pos 1177:21K pos 2.612 pos 3.049 pos 1.077 pos1177:63O pos 0.290 pos 2.091 pos 0.215 pos 3818:57A neg 0.039 neg 0.037neg 0.041 neg 3818:57C neg 0.038 neg 0.036 neg 0.042 neg 3818:57D neg0.037 neg 0.037 neg 0.040 neg

Example 12 Assay Results for Sera from 6 Dogs Experimentally Infectedwith E. Canis Using Synthetic Peptide (SEQ ID NO:23) Coated MicrotiterPlates and Anti-Canine Conjugate as Indicator

Six naïve dogs were experimentally infected with the Louisiana isolateof E. canis. Serum samples were obtained on days 3, 7, 10, 13, 17, 21,24, 28 and 35 post infection. Samples were tested using microtiter-platebased immunoassays prepared using the p16-2 synthetic peptide (SEQ IDNO:23) derived from the E. canis p16 surface protein. The syntheticpeptide was immobilized in microtiter wells, a dilution of the testsample (1:100) was added to the microtiter well and unbound antibody wasremoved by washing. Antibody bound to the immobilized peptide wasdetected by reaction with an anti-canine, horseradish peroxidase (HRPO)conjugate (1:2000 dilution), washing and addition of HRPO substrate. Theoptical density of individual microtiter wells was determined using amicrotiter plate reader.

Results:

Assay results are shown in Table 9. All 6 dogs converted from a negativestatus to a positive status following experimental infection as measuredby the commercially available reversible flow chromatographic SNAP® 4Dx®assay (reversible flow chromatographic assay). Sera from all dogsreacted to the E. canis p16-peptide shown in SEQ ID NO:23 at varioustimes post infection. The times between experimental infection andinitial reaction to the SEQ ID NO:23 peptide were as follows: Dog108532, 17 days post-infection; Dog 115853, 13 days post-infection; Dog265006, 17 days post-infection; Dog 268830, 13 days post-infection; Dog285307, 13 days post-infection and Dog533573, 13 days post-infection.

Conclusions:

The results demonstrate that antibody induced as a result ofexperimental infection was reactive to the synthetic peptide derivedfrom the E. canis p16 protein (SEQ ID NO:23).

TABLE 9 Assay results using for serum from dogs experimentally infectedusing E. canis synthetic peptide (SEQ ID NO: 23) coated microtiterplates and anti-species conjugate as indicator. A650 is absorbance at650 nm. The “4Dx ® SNAP ®” (reversible flow chromatographic assay)columnpresents the results from the reversible flow chromatographic SNAP ®4Dx ® assay (reversible flow chromatographic assay). 4Dx SEQ ID TimeSNAP NO: 23 Canine Sample Point EC A650 Result 1049:16E PC 2.253 +21172M NC 0.035 N Cutoff 0.070 108532 E1-0 d3 Neg 0.034 N E1-1 d7 Neg0.035 N E1-2 d10 Neg 0.037 N E1-3 d13 Neg 0.069 N E1-4 d17 Neg 1.501 +E1-5 d21 Neg 1.662 + E1-6 d24 + (.04) 1.572 + E1-7 d28 + (.06) 1.604 +E1-8 d35 + (.10) 2.056 + 115853 E2-0 d3 Neg 0.034 N E2-1 d7 Neg 0.033 NE2-2 d10 Neg 0.039 N E2-3 d13 Neg 1.246 + E2-4 d17 Neg 1.393 + E2-5 d21Neg 1.227 + E2-6 d24 + (.04) 1.549 + E2-7 d28 + (.03) 1.580 + E2-8 d35 +(.04) 1.939 + 265006 E3-0 d3 Neg 0.042 N E3-1 d7 Neg 0.035 N E3-2 d10Neg 0.038 N E3-3 d13 Neg 0.052 N E3-4 d17 Neg 0.944 + E3-5 d21 Neg1.031 + E3-6 d24 Neg 0.962 + E3-7 d28 Neg 0.840 + E3-8 d35 + (.05)1.303 + 268830 E4-0 d3 Neg 0.037 N E4-1 d7 Neg 0.034 N E4-2 d10 Neg0.038 N E4-3 d13 Neg 0.112 + E4-4 d17 Neg 1.432 + E4-5 d21 + (.05)1.364 + E4-6 d24 + (.05) 1.167 + E4-7 d28 + (.09) 1.412 + E4-8 d35 +(.12) 1.986 + 285307 E5-0 d3 Neg 0.036 N E5-1 d7 Neg 0.046 N E5-2 d10Neg 0.044 N E5-3 d13 Neg 1.018 + E5-4 d17 Neg 1.597 + E5-5 d21 + (.05)1.478 + E5-6 d24 + (.04) 1.282 + E5-7 d28 + (.04) 1.329 + E5-8 d35 +(.10) 1.838 + 533573 E6-0 d3 Neg 0.037 N E6-1 d7 Neg 0.035 N E6-2 d10Neg 0.032 N E6-3 d13 Neg 0.909 + E6-4 d17 Neg 1.832 + E6-5 d21 + (.08)1.883 + E6-6 d24 + (.08) 1.964 + E6-7 d28 + (.06) 1.963 + E6-8 d35 +(.15) 2.166 +

Example 13 Preparation of Formalin Inactivated E. Canis for Immunizationinto Dogs

E. canis was grown in canine cell culture using methods described in theliterature. See Breitschwerdt, Antimicrobial Agents and Chemotherapy,1998, Vol 42:362-368. Using light microscopy, 030 cells were estimatedto be greater than 80% infected by E. canis. Two liters of E. canisinfected cell culture were collected, centrifuged and the pelletretained yielded 7.31 gms of material (wet weight). It is presumed watermade up 80% of the weight of the material, giving an estimated dryweight of 1.462 gms (20% of the weight of the material). The cell pelletwas resuspended to 20 mg/ml in PBS (dry weight) for a total volume of 73ml.

To this resuspended cell pellet, 0.73 ml of formalin solution was added(Sigma Catalog HT50-1-2 Formalin Solution 10%, neutral buffered) for afinal formaldehyde concentration of 0.04%. The solution was stirred 12to 24 hrs at 4° C. The inactivated mixture was centrifuged and the cellpellet retained. The pellet was washed by resuspension into 250 mls ofPBS. The material was collected by centrifugation and the wash wasrepeated one time.

The sample was aliquoted to 73 screw cap vials and frozen at −80° C.Each vial contains 20 mgs (dry weight) of formalin inactivated E. caniscell culture, suitable for combining with the appropriate adjuvant forimmunization into animals.

Example 14 Preparation of Formalin Inactivated E. Canis with TwoDifferent Adjuvants and Protocol for the Immunization of Beagles with E.Canis Antigen

For immunization into kennel-housed dogs (laboratory beagles) formalininactivated E. canis antigen was formulated using three differentadjuvants. Formalin inactivated E. canis antigen was prepared with Ribiadjuvant (Corixa Corp., Seattle Wash.) using the protocol described bythe manufacturer. Each dose contained approximately 20 mg of formalininactivated E. canis cell culture (dry weight). An additionalformulation of immunogen was prepared using a combination of the Ribiadjuvant (described above) and the adjuvant BCG (1 mg per dose)(Calbiochem of EMD Biosciences, Inc., San Diego, Calif.). Two groupsconsisting of three dogs each were dosed 4 times over a period of 170days (days 0, 14, 156, 170) using inactivated E. canis containing eitherRibi adjuvant alone or Ribi adjuvant and BCG adjuvant in combination. Inan effort to produce a vaccine-induced hyperimmune state, all dogsreceived a single dose (day 247) of formalin inactivated E. canisformulated using the adjuvant TiterMax® (CytRx Corp., Norcross, Ga.) orthe adjuvant TiterMax® and the adjuvant BCG in combination using themanufacturer's instructions. Dogs were administered vaccines accordingto the following schedule:

Vaccine Administered/Day of Vaccination E. canis/ E. canis/ E. canis/TiterMax + Dog ID E. canis/Ribi Ribi + BCG TiterMax BCG CVYDEH 0, 14,156, 247 170 CWMBDC 0, 14, 156, 247 170 CVXCSM 0, 14, 156, 247 170CWMAXK 0, 14, 156, 247 170 CVSCVA 0, 14, 156, 247 170 CVXCAP 0, 14, 156,247 170

The IACUC committee of Covance Research Products Inc. approved theprotocol for immunization of laboratory beagles. All dogs were dosedwith the appropriate test article subcutaneously in the dorsoscapulararea. On day 0 all 6 dogs were found to be sero-negative using both thereversible flow chromatographic SNAP® 3Dx® diagnostic (reversible flowchromatographic assay) as well as western blot analysis using E. canisorganism. All six animals seroconverted to a positive test on thereversible flow chromatographic SNAP®3Dx® E. canis assay (reversibleflow chromatographic assay) by day 42. Production bleeds were taken ondays 226, 261, 268 and 282. (approximately 50 ml blood that yieldedapproximately 25 ml sera).

Example 15 Testing of Sera from Beagles Immunized with FormalinInactivated E. Canis (Vaccine Samples) Using a Microtiter-Plate BasedImmunoassay Prepared Using the Synthetic Peptides (SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24) Obtained from the E. Canis Protein p16 Protein

Preparation of the formalin inactivated E. canis and immunization ofbeagles were described in Examples 13 and 14. Samples from immunizedbeagles were tested using the direct microtiter-plate based immunoassaysprepared using the synthetic peptides (SEQ ID NO:22, SEQ ID NO:23 andSEQ ID NO:24).

Direct Assay Format

Samples were tested using microtiter-plate based immunoassays preparedusing the synthetic peptides (SEQ ID NO:22, SEQ ID NO:23 and SEQ IDNO:24). The synthetic peptides were immobilized on microtiter platewells at 1.0 ug/ml. Separate quantities of the synthetic peptides wereconjugated to the indicator reagent horseradish peroxidase (HRPO). Thetest sample and the immunoassay peptide/indicator were added to thepeptide-coated microtiter well, which was incubated and washed. Antibodybound to the immobilized peptide and the peptide/indicator reagent wasimmobilized in the microtiter well. This complex was detected byaddition of an HRPO substrate reagent. The optical density of individualmicrotiter wells was determined using a microtiter plate reader.

Results

Assay results are shown in Table 10. The positive control (PC, ID1049:16E) and negative control (NC, 3818:57B) were known E. canispositive and negative serum samples, respectively. All samples weretested using the commercially available the reversible flowchromatographic SNAP® 4Dx® test (reversible flow chromatographic assay)for E. canis antibody. Results for sequential temporal samples from the6 dogs (CVYDEH, CWMBDC, CVXCSM, CWMAXK, CVSCVA and CVXCAP) receiving theformalin inactivated E. canis antigen formulated using differentadjuvants are shown for day 226, day 261, day 268 and day 282post-immunization. Results of the reversible flow chromatographic SNAP®4Dx® test demonstrate that an antibody response was induced in thevaccinated animals. None of the serum samples from vaccinated animalswas reactive in the peptide (SEQ ID NO:22, SEQ ID NO:23 and SEQ IDNO:24) microtiter plate-format assay.

TABLE 10 Assay results using for serum from dogs immunized with formalininactivated E. canis antigen measured using E. canis synthetic peptide(SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24) coated microtiter platessynthetic peptide- conjugate as indicator. OD is optical density. The“4Dx ® SNAP ®” (reversible flow chromatographic assay) column presentsthe results from the reversible flow chromatographic SNAP ® 4Dx ® assay(reversible flow chromatographic assay). Plate Results SEQ ID SEQ ID SEQID 4Dx SNAP NO: 22 NO: 23 NO: 24 Adjuvant Sample OD/(result) OD RESULTOD RESULT OD RESULT 1049:16E (PC) 0.72 2.276 2.865 1.021 3818:57B (NC)neg 0.043 neg 0.044 neg 0.042 neg Ribi CVYDEH day neg 0.038 neg 0.056neg 0.037 neg 226 day 261 0.07 (pos) 0.037 neg 0.046 neg 0.035 neg day268 0.17 (pos) 0.045 neg 0.057 neg 0.038 neg day 282 0.18 (pos) 0.035neg 0.048 neg 0.034 neg Ribi CWMBDC day 0.08 (pos) 0.050 neg 0.052 neg0.043 neg 226 day 261 0.45 (pos) 0.044 neg 0.090 neg 0.039 neg day 2680.40 (pos) 0.039 neg 0.064 neg 0.038 neg day 282 0.30 (pos) 0.038 neg0.058 neg 0.040 neg Ribi CVXCSM neg 0.034 neg 0.038 neg 0.042 neg day226day 261 neg 0.044 neg 0.073 neg 0.071 neg day 268 0.14 (pos) 0.042 neg0.038 neg 0.041 neg day 282 0.23 (pos) 0.044 neg 0.038 neg 0.054 negRibi + BCG CWMAXK day 0.07 (pos) 0.038 neg 0.035 neg 0.039 neg 226 day261 0.26 (pos) 0.043 neg 0.037 neg 0.036 neg day 268 0.36 (pos) 0.045neg 0.043 neg 0.034 neg day 282 0.34 (pos) 0.038 neg 0.036 neg 0.034 negRibi + BCG CVSCVA day  .10 (pos) 0.039 neg 0.036 neg 0.041 neg 226 day261 0.51 (pos) 0.041 neg 0.036 neg 0.036 neg day 268 0.45 (pos) 0.043neg 0.035 neg 0.035 neg day 282 0.47 (pos) 0.036 neg 0.037 neg 0.036 negRibi + BCG CVXCAP day neg 0.041 neg 0.036 neg 0.034 neg 226 day 261 0.51(pos) 0.054 neg 0.046 neg 0.047 neg day 268 0.42 (pos) 0.041 neg 0.045neg 0.043 neg day 282 0.48 (pos) 0.035 neg 0.034 neg 0.036 negConclusions

The results demonstrate that antibodies induced as a result ofimmunization using formalin inactivated E. canis antigen were reactiveon the reversible flow chromatographic SNAP® 4Dx® test (reversible flowchromatographic assay) which would indicate that an anti-E. canisantibody response was initiated. These same samples were nonreactive tothe synthetic peptides derived from the E. canis P16 protein (SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24).

Sera from dogs immunized with formalin inactivated E. canis antigen werenonreactive to the peptides derived from the E. canis P16 protein (SEQID NO:22, SEQ ID NO:23, SEQ ID NO:24). The synthetic peptides (SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24) were nonreactive to antibody inducedas a result of vaccination.

Example 15 Monitoring Treatment of E. Canis Infection

Six dogs were experimentally infected with E. canis. Doxycycline wasadministered at 28 days post-infection. Antibodies specific for E. caniswere detected using SEQ ID NO:23 with an indirect assay protocol.Polypeptides shown in SEQ ID NO:23 were immobilized on microtiter wellsby direct adsorption. A dilution of the test sample (1:100) was added tothe microtiter well and unbound antibody was removed by washing.Antibody bound to the immobilized peptide was detected by reaction withan anti-species, in this case rabbit anti-canine horseradish peroxidase(HRPO) conjugate (1:1000 dilution). The absorbance (A650) of individualmicrotiter wells was determined using a microtiter plate reader. Thenegative cutoff was 2× the negative control O.D. value.

The results are shown in FIG. 8. The E-1, E-2, E-3, E-4, E-5, and E-6dogs were experimentally infected with E. canis, but were not treatedfor the infection. FIG. 8 demonstrates that the level of antibodies thatbind to SEQ ID NO:23 increased considerably after experimental infectionand did not decrease during the time course of the experiment. TheEDTx-1, EDTx-2, EDTx-3, EDTx-4, EDTx-5, and EDTx-6 dogs wereexperimentally infected with E. canis and then treated with doxycyclineat 28 days-post infection. FIG. 8 demonstrates that the level ofantibodies that bind to SEQ ID NO:23 increased considerably afterexperimental infection and decreased after administration ofdoxycycline. Therefore, SEQ ID NO:23 can be used to monitor theprogression, the response to treatment, or the efficacy of treatment ofE. canis infection.

Example 16 Differentiation of Dogs that have been Vaccinated for E.Canis and Dogs that have been Vaccinated for E. Canis, but have BecomeInfected with E. Canis

Vaccines may not be completely effective at preventing infection.Therefore, it is desirable to have a method to determine if a vaccinatedanimal has become infected despite the vaccination. Immunoassays usingp16 as a detection agent do not detect anti-E. canis antibodies in dogsthat have been vaccinated for E. canis and that are not infected with E.canis. It has now been discovered that an E. canis p16 protein (SEQ IDNO:10) can be used to detect E. canis infection in dogs that havereceived an E. canis vaccine.

Six dogs that had been vaccinated for E. canis and two unvaccinated dogswere challenged with E. canis infected K9 cells in 10% DMSO. Each dogwas tested over time for anti-E. canis antibodies with an immunoassaycomprising SEQ ID NO:10. All of the vaccinated dogs and the two controldogs became infected with E. canis. The E. canis infections wereconfirmed with two independent infection markers. The immunoassays wereable to detect the E. canis infection in the vaccinated andnon-vaccinated dogs. All of the immunoassay signals were significantlyabove background signals. See FIGS. 9A-B, 10A-C, and 11A-C.

Sequences:

SEQ ID NO: 1 120 kDa Antigen Nucleotide Sequence ORIGIN 1ATGGATATTG ATAACAATAA TGTGACTACA TCAAGTACGC AAGATAAAAG TGGGAATTTA 61ATGGAAGTGA TTATGCGTAT ATTAAATTTT GGTAATAATT CAGATGAGAA AGTAAGCAAT 121GAAGACACTA AAGTTCTTGT AGAGAGTTTA CAACCTGCTG TGAATGACAA TGTAGGAAAT 181CCATCAAGTG AAGTTGGTAA AGAAGAAAAT GCTCCTGAAG TTAAAGCGGA AGATTTGCAA 241CCTGCTGTAG ATGGTAGTGT AGAACATTCA TCAAGTGAAG TTGGGAAAAA AGTATCTGAA 301ACTAGTAAAG AGGAAAGTAC TCCTGAAGTT AAAGCAGAAG ATTTGCAACC TGCTGTAGAT 361GGTAGTATAG AACATTCATC AAGTGAAGTT GGAGAAAAAG TATCTAAAAC TAGTAAAGAG 421GAAAGTACTC CTGAAGTTAA AGCAGAAGAT TTGCAACCTG CTGTAGATGA TAGTGTGGAA 481CATTCATCAA GTGAAGTTGG AGAAAAAGTA TCTGAAACTA GTAAAGAGGA AAATACTCCT 541GAAGTTAAAG CAGAAGATTT GCAACCTGCT GTAGATGGTA GTATAGAACA TTCATCAAGT 601GAAGTTGGAG AAAAAGTATC TAAAACTAGT AAAGAGGAAA GTACTCCTGA AGTTAAAGCA 661GAAGATTTGC AACCTGCTGT AGATGATAGT GTGGAACATT CATCAAGTGA AGTTGGAGAA 721AAAGTATCTG AAACTAGTAA AGAGGAAAAT ACTCCTGAAG TTAAAGCAGA AGATTTGCAA 781CCTGCTGTAG ATGGTAGTGT GGAACATTCA TCAAGTGAAG TTGGAGAAAA AGTATCTAAA 841ACTAGTAAAG AGGAAAGTAC TCCTGAAGTT AAAGCAGAAG ATTTGCAACC TGCTGTAGAT 901GATAGTGTGG AACATTCATC AAGTGAAGTT GGAGAAAAAG TATCTGAAAC TAGTAAAGAG 961GAAAATACTC CTGAAGTTAG AGCAGAAGAT TTGCAACCTG CTGTAGATGG TAGTGTAGAA 1021CATTCATCAA GTGAAGTTGG AGAAAAAGTA TCTGAAACTA GTAAAGAGGA AAGTACTCCT 1081GAAGTTAAAG CAGAAGATTT GCAACCTGCT GTAGATAGTA GTATAGAACA TTCATCAAGT 1141GAAGTTGGGA AAAAAGTATC TGAAACTAGT AAAGAGGAAA GTACTCCTGA AGTTAAAGCA 1201GAAGATTTGC AACCTGCTGT AGATGGTAGT GTAGAACATT CATCAAGTGA AGTTGGAGAA 1261AAAGTATCTG AAACTAGTAA AGAGGAAAAT ACTCCTGAAG TTAAAGCAGA AGATTTGCAA 1321CCTGCTGTAG ATGGTAGTGT AGAACATTCA TCAAGTGAAG TTGGAGAAAA AGTATCTGAA 1381ACTAGTAAAG AGGAAAATAC TCCTGAAGTT AAAGCGGAAG ATTTGCAACC TGCTGTAGAT 1441GGTAGTGTAG AACATTCATC AAGTGAAGTT GGAGAAAAAG TATCTGAAAC TAGTAAAGAA 1501GAAAGTACTC CTGAAGTTAA AGCAGAAGAT TTGCAACCTG CTGTAGATGA TAGTGTAGAA 1561CATTCATCAA GTGAAGTTGG AGAAAAAGTA TCTGAAACTA GTAAAGAAGA AAGTACTCCT 1621GAAGTTAAAG CGGAAGATTT GCAACCTGCT GTAGATGGTA GTGTGGAACA TTCATCAAGT 1681GAAGTTGGAG AAAAAGTATC TGAGACTAGT AAAGAGGAAA GTACTCCTGA AGTTAAAGCG 1741GAAGTACAGC CTGTTGCAGA TGGTAATCCT GTTCCTTTAA ATCCTATGCC TTCAATTGAT 1801AATATTGATA CTAATATAAT ATTCCATTAC CATAAAGACT GTAAAAAAGG TTCAGCTGTA 1861GGAACAGATG AAATGTGTTG TCCTGTATCA GAATTAATGG CTGGGGAACA TGTTCATATG 1921TATGGAATTT ATGTCTATAG AGTTCAATCA GTAAAGGATT TAAGTGGTGT ATTTAATATA 1981GATCATTCTA CATGTGATTG TAATTTAGAT GTTTATTTTG TAGGATACAA TTCTTTTACT 2041AACAAAGAAA CAGTTGATTT AATATAASEQ ID NO: 2 120 kDa Antigen Protein Sequence ORIGIN 1MDIDNNNVTT SSTQDKSGNL MEVIMRILNF GNNSDEKVSN EDTKVLVESL QPAVNDNVGN 61PSSEVGKEEN APEVKAEDLQ PAVDGSVEHS SSEVGKKVSE TSKEESTPEV KAEDLQPAVD 121GSIEHSSSEV GEKVSKTSKE ESTPEVKAED LQPAVDDSVE HSSSEVGEKV SETSKEENTP 181EVKAEDLQPA VDGSIEHSSS EVGEKVSKTS KEESTPEVKA EDLQPAVDDS VEHSSSEVGE 241KVSETSKEEN TPEVKAEDLQ PAVDGSVEHS SSEVGEKVSK TSKEESTPEV KAEDLQPAVD 301DSVEHSSSEV GEKVSETSKE ENTPEVRAED LQPAVDGSVE HSSSEVGEKV SETSKEESTP 361EVKAEDLQPA VDSSIEHSSS EVGKKVSETS KEESTPEVKA EDLQPAVDGS VEHSSSEVGE 421KVSETSKEEN TPEVKAEDLQ PAVDGSVEHS SSEVGEKVSE TSKEENTPEV KAEDLQPAVD 481GSVEHSSSEV GEKVSETSKE ESTPEVKAED LQPAVDDSVE HSSSEVGEKV SETSKEESTP 541EVKAEDLQPA VDGSVEHSSS EVGEKVSETS KEESTPEVKA EVQPVADGNP VPLNPMPSID 601NIDTNIIFHY HKDCKKGSAV GTDEMCCPVS ELMAGEHVHM YGIYVYRVQS VKDLSGVFNI 661DHSTCDCNLD VYFVGYNSFT NKETVDLI.SEQ ID NO 3 200 kDa Antigen nucleotide sequence from 1081 to end ORIGIN1  AATTTAGAT TTTGGACTTG TAGATGGAGA TGGTAAAAAT CCTTTACATC ATGCTGTTGA 61ACATTTGCCA CCTGTTATAC TTAAGGGCGT AATGGACCAT GTAAAAAATA GTAGTGAGTT 121TCAAGATTTA GTAAATGATC CTGATTATTT TGGAAATACT ATAGCTCATT ATGCAGTTAA 181GAATAAAAAT GCTGATTTAA CATTGTTTAA CATGCTGAAA GCTTCAGGAG CTGATTTAAA 241TGTTAGGAAT GTAGTTGGTC GAGCTCCAAT ACATGTTGCT TCTTCTAATG GTAAGGCTAA 301TGCAGTTTCT GGACTTGTAT CATGTGGTAT TGACGTTAAT TCTCAAGATG TGAATGGAGA 361TACACCACTT CATATTGCTG TTGAAGGCGG TAGTATGGAG ACGGTATTAG CAGTGTTAAA 421TCAGAGAGGT GCTGATGTTA GTGTCCAGAA TAACGATGGA GTTACACCTA TGCTTAGTGC 481TGCTAAATAT GGAGATATAG GTGTAATAAA AGCTTTAGGT TCAGCTAAAC CAAATATTAA 541AGGTGAAGAC ACTGTTGCTA AATCATTGCT GATGGAGGAT TACAAAGGTT TTACACCCTT 601GCATTTTGTA GCTGGTGGTG GTAGCAGAGA TACATTCCGT GTCGTAAGAA AAAATTATGA 661AAAATGTCAT GACTTAGCTA CTATTAGGGC AGCTTTAATG CAAGATAGAA GTGGTGGTGA 721GCTTGTAAAT TTAGGGGATT TTGAAAGTGA AAATATATTG GGTTCGCCAA ATGCAAAATT 781CTTGCAGCAT ATTCAATCAG CAAATTTTGG TTTTTCTCCA GCGCATTGTG CTATAGTATC 841GTCTAATCAC AATGTAATGA AAGATATCTT AAATTTTGTT GGGGATTCGT TACACCTACC 901AAGTGAGCGT GGGTATAATG CAATGCAGGT TGCTGCTTTG TTTGGTGACA AAGAAGCAGT 961GAAAATGCTT GCTAAAAGTG CTAAGCCAAG TGATCTTAAT TTTAAGACTT CAGCAACTCC 1021TACTCCGTTA AATCTTGCAT GTCTTAGAGG TGATAATGAG GTAGTACGTG GGTTAGTAGG 1081TCAACATGGT ATTGACATTA ACCAACGTAT GGGAAGTGAT AAAAACACTG TATTGCATTA 1141TGCAATCAGC AAAGGAGATA GTTTTCTTGT GCAAAAGATA TTAGCTCATA CTGGAGTTGA 1201TGTTAATTGT GAGAATAACC TAGGTCAAAC GCCTTTACAT TTAGCAGTTG AGGGAGGAGA 1261TCCTAAGATA GTATCTTCTC TTCTTAAAGC TGGTGCAGTA GTTAATCGTC TGGATGATAA 1321TGGTAGATCT GTACTTTCTT CTGCGATAGT TCCAGGTAGA AAAGAAAAGG GAGTGCTGGG 1381TATAGTTAAT AAATTGCTGG ATAGAGGTGC AGATATTAAT TTAGATGGAG ACCACAATAT 1441ACTTTTTGAT CAGTGTCTAA GGGGTGGATA TAATAATGTA TTAGATAAGT TAATACAACA 1501AGGGGTTGAA GTTAATCGAA ATAGTGAAAT ACGTCCAATG GTTTATGCTG CAATATCTGG 1561TAATGAGCAT GCTATCAAAT CATTAGCTAA TGCTGGTGGA GATGTTAATG AAGTAGTAAA 1621TAATCCATCT AGTAGGCATT CAGGAAATCC TTTAATTATG GTTGCAGTAG CAGATGGTAA 1681TGCAGGTCTT CTTAAAACAT TAGTTTCTGA AGGATGTGAT GTTGGTAAAT CTGGAAAAGA 1741TGGTAATACA GCGTTACATT ATGCTGTTAG TCATTCAGAT AAAGAGTTTG GTAATAAAGC 1801TATAAAGATA TTAATTTCAC GTAATAGTGT TGGGACTAAT AGAGATATTC TTACTCAAAA 1861GAATAACGCA GGTGATACAC CTTTACATGA AGCTCTTAAG TCAGGTAATA TTAATTCTGT 1921ACAGAATATC TTAAGTGCTG TACATCCAAG ATACGCAAAG GAGATATTAA CAGCCAGAGA 1981CAAAGAAGGG TACACACCAA TGCATTATAC TGTTGGAGTA AATAATGTTG ATGTTGGTAG 2041AAGTATTCTA GAGTCTATGC TCTCTAAAGG TGTGAATAAT CTTGGAGAGA TTGTTGGAGC 2101ACAGGATAGT AATTTTCGAA CACCTCTGCA TGCTGCTATT AAAATATCTG ATTATCGTGC 2161TGCGGACATG ATAATAGGTA GCTTATCGAA AACAGAATTG TCAAAGTTAT CGCAATTAAC 2221AGATATTAAC GGGGATACAC CACTACATCT TTCTTGTCAG TCTGGTAATG TCGAGATGAC 2281ACAATTCTTT CTTGGAGGTT TGGATAAACG TGAATTACCT AAGACATTAA AGATAGCAAA 2341TAAAAATGGA GATACTCCTT TACATGATGC TATAAGAAAT GATGATATTA AATCTGCAAA 2401AATGATGATT AGGAATTGTA ACAAAGAAGA ACTTGCTAAT GTATTAAAAT GTAAAGATAG 2461TTTTGGTAAT ACAGTATTGC ATACTATTGC TGACCAAGTT ATTGCGAATC CAGAATCAAA 2521GAAAGACCTT GATGGTTTGA TGAATTTAGC AGTGAAAAGG CTAAAGAATC AAGATCTGAA 2581AGATCTAGTT AATACGCGAA ATAACTCTGA CGATACTGTT GCACATTGTG CTCTTTTATC 2641GGATATGAAA TATGCTCAAA AGATACTTAA ATCATGTAAC CATGATACAT TAGTGAGAGG 2701AAATAGTAAT AATCAATCTT TATCAGAGTG TATTCGTGAT GATAGTAAAT ATAAAAAAGG 2761TGGAATTTTT AGTAAGTCTT TATTTTCAAA ATTAAAGAAA CTTGAGGCAC GAGCTGCCAG 2821CGCTAGTTAT GAAGAATTAT CTAGTATCAG TAGTGGTAGT GATGTTTCTT CTGTATCAAC 2881AAATAGCACA GAAGTAAGTG CAGTACCTGA AGTGGCAAGA AGTAGTGGTG CTGTGTCGTT 2941CAAACATGTG CAAGAAACAG GAGTTGACAC GTCTGGTCCT TCTGATATAG AAAGTTTAGA 3001GAGATTATCT GATACTAGTC TTGGGTCAAA TGATTTTGAT CAGCGAATGG CAGATTTAGA 3061TCAAGAAATA GCAAATATTG TTAGTGGTTT ACCAGAAGTT ACCCAGGTAG CTGTAAGTCA 3121ACAACAAGCA GCATCTCCTA GTTCAGGTCA AGCTGCTGGT GTGCAACAAA AAGAGATGCA 3181GAGATAA SEQ ID NO: 4 200 kDa Antigen Partial Protein Sequence ORIGIN 1NLDFGLVDGD GKNPLHHAVE HLPPVILKGV MDHVKNSSEF QDLVNDPDYF GNTIAHYAVK 61NKNADLTLFN MLKASGADLN VRNVVGRAPI HVASSNGKAN AVSGLVSCGI DVNSQDVNGD 121TPLHIAVEGG SMETVLAVLN QRGADVSVQN NDGVTPMLSA AKYGDIGVIK ALGSAKPNIK 181GEDTVAKSLL MEDYKGFTPL HFVAGGGSRD TFRVVRKNYE KCHDLATIRA ALMQDRSGGE 241LVNLGDFESE NILGSPNAKF LQHIQSANFG FSPAHCAIVS SNHNVMKDIL NFVGDSLHLP 301SERGYNAMQV AALFGDKEAV KMLAKSAKPS DLNFKTSATP TPLNLACLRG DNEVVRGLVG 361QHGIDINQRM GSDKNTVLHY AISKGDSFLV QKILAHTGVD VNCENNLGQT PLHLAVEGGD 421PKIVSSLLKA GAVVNRLDDN GRSVLSSAIV PGRKEKGVLG IVNKLLDRGA DINLDGDHNI 481LFDQCLRGGY NNVLDKLIQQ GVEVNRNSEI RPMVYAAISG NEHAIKSLAN AGGDVNEVVN 541NPSSRHSGNP LIMVAVADGN AGLLKTLVSE GCDVGKSGKD GNTALHYAVS HSDKEFGNKA 601IKILISRNSV GTNRDILTQK NNAGDTPLHE ALKSGNINSV QNILSAVHPR YAKEILTARD 661KEGYTPMHYT VGVNNVDVGR SILESMLSKG VNNLGEIVGA QDSNFRTPLH AAIKISDYRA 721ADMIIGSLSK TELSKLSQLT DINGDTPLHL SCQSGNVEMT QFFLGGLDKR ELPKTLKIAN 781KNGDTPLHDA IRNDDIKSAK MMIRNCNKEE LANVLKCKDS FGNTVLHTIA DQVIANPESK 841KDLDGLMNLA VKRLKNQDLK DLVNTRNNSD DTVAHCALLS DMKYAQKILK SCNHDTLVRG 901NSNNQSLSEC IRDDSKYKKG GIFSKSLFSK LKKLEARAAS ASYEELSSIS SGSDVSSVST 961NSTEVSAVPE VARSSGAVSF KHVQETGVDT SGPSDIESLE RLSDTSLGSN DFDQRMADLD 1021QEIANIVSGL PEVTQVAVSQ QQAASPSSGQ AAGVQQKEMQ R.SEQ ID NO: 5 ATPase - Clone 84 Fragment Nucleotide Sequence ORIGIN 1AATTATGCTG AAACTACTTT ATCATTTGGT GAATCTCGAG CAGAAGGACG TGAATCTCCA 61TCAAGTGCAT TTGTTCAAAC TGGTCAATCA GAAGTACCTC GGAGTGAGGC TGCAGAGCCA 121TTAATTCAAT TTCCTCATGA TGAAGAAAGT ACTGCATTAG GTTCTCAAGC AACTATGACA 181GGAGTGTCTA CTCAGGCTAG TCCGTCAGCA GCATATCAGG ATGATAGTGA AATATCACGT 241ATGAGGTCTA TGGCAGGAAC ATCTGCTCAA GCTGATCAAT CAGCAGTACA TCGTCGGAGT 301GGTACAGCAT TAGAGCCATT AATTGAATTG CCTGATGAAG AAGAAAATGC TGCATTAGAT 361TTTCAAACAG CTATGACAGG AGTGCCTACT CAGGCTAGTC CGTCAGCAGT ACATCGGAGT 421GGTGTTGCAT CAGATCCTAC GCTACCTGAT GATGAAAGAA TTGATGTTCC ATCAGTTTCA 481TCTCAAGTTG TAAGACCTTT TAGTGATGGT GAAGATTATT CAGTATATGA TAAATCAGGT 541GTAGTAAGTG GTCATGAAAG ACCTGTTTCT TCTAGAGATT CAAGACAATT GGATGCATTT 601GGTGATCCAT CAGATGATTT ATTGCCGGAG AGTGAAATTA TTGTTAGCAG CAGTAAGAAA 661GCAATATTAG ATAGCCAAAA TGAAATAGAA TCTCTTATTC AGAGTGGAGA TACTTCTAGA 721TGTATTAGGG CAATTAATAG TGCTCCTAGT GCGTCAGTGT TTCAACTGAA GACTTTATCG 781AATGATATAT CTATTGCTGG ACGTGCTTTT TTAAATGGTA ATATTGATTT AATAGAAGCT 841TGTATGAATT CTGGCAAGAA ATTAAATCCA AATATTACTG ATAATGAAAA AAATACTCTA 901TTACATCAAT TTGTAGGATA TTTTGAACGC GATCCGAGAA TGTTGCTTGA TGCAGGAATG 961CGTAATCTGT TTTTGAGATT ATGCATGGAT TATGGTTTCG ATATTAATCA TAAAAATAGT 1021AATGGTAATA CAGTACTTGA TAGATTAAAT GATTTAGTAG AAGGGTTAAG TAGTTCGCAA 1081GTTGATCTTG AAAGTAGTGG TATTGATGAG TTTATGATCT CATTGTTAGC TCATTCTAGA 1141ATGAGTGATC AAGCAGTAAA GAATATTGCT ACTGCGCAAA ATGAGTTTTT TGCACGTGAT 1201TCTGTTTATA ATATTAGTCG TTTAGTTGAT ACTTCTATAG TTTTGCAGAA TAAATTCAGT 1261GAAGTATTTT ATGAAGTCTG TGGACGTATT TTATCTGAAG AAGCTGGTAA ACATAAGGGT 1321GTTGCTGAAG CAAATTATTC AAGATTGAAT AAAATATTAA ATGATGAATG TCTTAGAAAG 1381ACTTTAGCTA ATACAGATGC CGATGGAAAT AATGTTTTAC AGAGATTGTG TCAAGATATT 1441GCTTCTGGAA AAATCAATGC TCGTGATGAC AGAGTATTAA AACTTTTTGA GACAATTATA 1501TCTAATTTAA AAGACAAAGA TAAAGCATTA CTAGAGGATT TATTATTTAA TAATAGAAAC 1561TCAAGATTTG AAAATTGCAT TGAAGCTATA CCACGTATTC CTGGTGCCGA TGCTCTATTT 1621AAAAAACTAG AAGAGTTATT ATTAAAAAAG AAAATAGCAG AGTCTTGTGA TTTTAATTCT 1681ATGTTAGTGA ATTGTGCTGA GTCTGCTAAT GATAATTTAT ATAATTACCT GCGCACTAAT 1741TATGCAGTTA TTGGTATAAA TAACGTAGAT ATAAATGGCA ATTCATCCCT ATGTAAAGCT 1801GTTGTTACTG GGTCACAAGG TATTGTTAAA GCAGTATTAT CAACTGGAAC TAATATTAAT 1861AGGAAAGATA AAAATGGTAA TACACCTTTA CATGCATTGT TAATTTTTAT GATGTCTAAC 1921CCTGAACTTG TCAAGGAGCA ACATATTTCA CTTGTGAAAT TCTTAGCGTC TCGTGGAGCT 1981TTACTTAATG TAAAAAATAA TATGAATATT TCTCCAATTA TGCTTGCAGA ATCTATTGAT 2041AAGAAAGAGG AACTTGCTAA GAAATTTACA AATCAAAAAG TTAGTATTTT AGAATCTTTA 2101ATAGCTGGTA GTGAAGAACA TTTAGGGCTT AAATCCAAAT GTATATCTGA GTTAAAGCCT 2161TATATAGAAT TAGGAAAAGG CATGAAGTAC GAAGATATAC ATGCTGATGT AATAGGTGGT 2221GTATTATCTG CTGATATGTG TAATGCTAGA TTGCAGATAG GTAAATTATT AAATGGTGAT 2281TTTTGTAAAG AAAATGAATT AAAGACAGTA AAATTTAATT TTTCTGATAC AAATAAGGGT 2341TATGTACAAA ATGTTGGTAA AAAAAGAAAT TATSEQ ID NO: 6 ATPase - Clone 84 Fragment Protein Sequence ORIGIN 1NYAETTLSFG ESRAEGRESP SSAFVQTGQS EVPRSEAAEP LIQFPHDEES TALGSQATMT 61GVSTQASPSA AYQDDSEISR MRSMAGTSAQ ADQSAVHRRS GTALEPLIEL PDEEENAALD 121FQTAMTGVPT QASPSAVHRS GVASDPTLPD DERIDVPSVS SQVVRPFSDG EDYSVYDKSG 181VVSGHERPVS SRDSRQLDAF GDPSDDLLPE SEIIVSSSKK AILDSQNEIE SLIQSGDTSR 241CIRAINSAPS ASVFQLKTLS NDISIAGRAF LNGNIDLIEA CMNSGKKLNP NITDNEKNTL 301LHQFVGYFER DPRMLLDAGM RNLFLRLCMD YGFDINHKNS NGNTVLDRLN DLVEGLSSSQ 361VDLESSGIDE FMISLLAHSR MSDQAVKNIA TAQNEFFARD SVYNISRLVD TSIVLQNKFS 421EVFYEVCGRI LSEEAGKHKG VAEANYSRLN KILNDECLRK TLANTDADGN NVLQRLCQDI 481ASGKINARDD RVLKLFETII SNLKDKDKAL LEDLLFNNRN SRFENCIEAI PRIPGADALF 541KKLEELLLKK KIAESCDFNS MLVNCAESAN DNLYNYLRTN YAVIGINNVD INGNSSLCKA 601VVTGSQGIVK AVLSTGTNIN RKDKNGNTPL HALLIFMMSN PELVKEQHIS LVKFLASRGA 661LLNVKNNMNI SPIMLAESID KKEELAKKFT NQKVSILESL IAGSEEHLGL KSKCISELKP 721YIELGKGMKY EDIHADVIGG VLSADMCNAR LQIGKLLNGD FCKENELKTV KFNFSDTNKG 781YVQNVGKKRN Y SEQ ID NO: 7 ATPase - Clone 7 Fragment Nucleotide SequenceORIGIN 1GTAAAAAAAT TAAGATTATT ATTAAATTCA ATAAGTGAGT TACCGCAAGA ATTAAAAGAT 61CAAATTTTAA GTACTAGAAG TACTATAGAT AAATTACGAA ATAGAATTAA TGCCTGCATA 121AAGTCTGACG ATAGAGAAGG TATTGCACAT GCTGTAGAAT CTATGGCTAG TTCTTATTGT 181GAATTATTAG GACATTGTAG ATTAATTTTT AAGAAATTAT ATGATGAAAA TGCTGATAAA 241AGTTTGCTAG AATTATGTAT TAAAGAATAT CAATCTGATT TAAACAAATT ATTGGAACAA 301GGTATTGATA TATGTGCTTC AGAAGTCTCA TCAGAATGTA AGGATTTAGT TTGTAAAGTA 361TGTGAAGATG AATTTGAGAA ATATGACTCT TTATCTAAAG TACAAAGATT CAGGGAATTA 421TCTGGTGAAA TTGCTGATTT GGATGATAAA TTAACAAGAA GGGCTTCTTT TGTTGAGACT 481TTTGGATTAT TTAGCAGTAG ATTAAGACAT TATAGGGAAA TTTTAGGAGA TGGTGATTTA 541AAATTTCGAG AGAGGATAGT TGAAAAATAT CAAGAGGATT TAAAGGAATT ATTAGAATTA 601TCTGTTGATC TTCATTTGTT AATAAATTTA CCAGCATTAG AAGATTTACG CGATCATAGA 661AATTTAGTGC ATAGAGCATG TAATGCTGAA ATTGAAAAAT ATCTAACTTT ATTTGATGAT 721CAACAATTAC GTACATTATC GCAAGAAGTG AATAATGCTC ATGGTGAATT GATACAGATG 781TTTTCTAAGT TTAGTATATT TGTTGATGGC GTTACTGGTA TTGAACAGAG CACATCTCAA 841GTAGAGCACC CTCGTTCTGA TATTGCTAAA AGAGATACTA CAACACCAAA GCAACGTGTT 901GTGCAAGGTA AAGATGATAT ACAATCTAGT GATAGTGATA GTGATAGTGA TAGTAAATAC 961GGTGATGATG ATAGTAAAAA AGCATCAGTT AGTGCACCTG CTGTTGACCA AGTTGTACCT 1021GTAGCTGATG TTCAACCTGA ACCTCAGCTA GGTGAAGGAT TGGAAACATT AGAGTCTAGT 1081ATAGCTGAAG GACCTGAGTT GCCTGGTGAT GCATCTACTG CTAAGCAATC TATACCTTTT 1141GCGATAACAC CATCAAGTCC TGAGACAGTT GATGAAAAAC TTGAAAGTTC TGGTGTTAGT 1201CAAGATGGTA TTACAACACC AGGACAACGT GTTGTGCAAG GTAAAGATGA TATACAATCT 1261AGTGATAGTG ATAGTGATAG TAAATACGGT GATGATGATA GTAAAAAAGC ATCAGCTAGT 1321GCACCTGCTG TTGACCAAGT TGTACCTGTA GCTGATGTTC AACCTGAACC TCAGCTAGGT 1381GAAAAATTGG AAACATTAGA GTCTAGTATA ACTAAAGGAC CTGAGTTGCC TGGTGATGCA 1441TCTACTGCTA AGCAATCTAT ACCTTTTGCG ATAACACCAT CAAGTCCTGA GACAGTTGAT 1501GAAAAACTTG AAAGTTCTGG TGTTAGTCAA GATGGTATTA CAACACCAGG ACAACGTGTT 1561GTGCAAGGTA AAGATGATAT ACAATCTAGT GATAGTGATA GTGATAGTAA ATACGGTGAT 1621GATGATAGTA AAAAAGCATC AGCTAGTGCA CCTGCTGTTG ACCAAGTTGT ACCTTCTGAC 1681ACTCGTGCAG ATGGAGTATC AGAACCATTA GCATCTCATG TGGATCAAGG ATCTGATGTA 1741CCTGGTGATG CATCTGTTGA TGGTGTTGAT TTAAGATTAG GACGGTTATC TACTGAGCAA 1801AGTGGATTGT TGCCACGTCA TGAACAAAAT GTAAGAGCAT TTATTTTAGA ACAGAGTTTG 1861TTAGATCAAT TATATATGGA CTATATAGAT TTACACCCTG ATCAGAAAAG TTGTGAAGCT 1921TATAATTCAG CATTGCATGG ATATAATACA AGATTAGAGT TACAGAAGGA ATATAACAGG 1981ATTTTTGAAT CACATGAATC AGCATCTCCA AATGAAATTA ATAGTTTTTC ACAAAAATAT 2041AGAGCAGCAT TAAGAGATGT TGCGCAGGAT ATTGTTAATC AGGGTCCAAT GTTTTATTCT 2101TCTAGAGATG CAATGCTATT AAGGGCTAGA GTAGACACAT TGTGTGATAT GTGTCGTTCA 2161ATACGTAATC TGTATATGGT TGAATTAGAT GCCATAGATA AAGAAGAAAA ATCGTTACAA 2221TCTGATATGA AATCTGCAAG TTCTAGTGAT AAAAAGTTGA TACAAGAAAA AATAAAATTA 2281CTT SEQ ID NO: 8 ATPase - Clone 7 Fragment Protein Sequence ORIGIN 1VKKLRLLLNS ISELPQELKD QILSTRSTID KLRNRINACI KSDDREGIAH AVESMASSYC 61ELLGHCRLIF KKLYDENADK SLLELCIKEY QSDLNKLLEQ GIDICASEVS SECKDLVCKV 121CEDEFEKYDS LSKVQRFREL SGEIADLDDK LTRRASFVET FGLFSSRLRH YREILGDGDL 181KFRERIVEKY QEDLKELLEL SVDLHLLINL PALEDLRDHR NLVHRACNAE IEKYLTLFDD 241QQLRTLSQEV NNAHGELIQM FSKFSIFVDG VTGIEQSTSQ VEHPRSDIAK RDTTTPKQRV 301VQGKDDIQSS DSDSDSDSKY GDDDSKKASV SAPAVDQVVP VADVQPEPQL GEGLETLESS 361IAEGPELPGD ASTAKQSIPF AITPSSPETV DEKLESSGVS QDGITTPGQR VVQGKDDIQS 421SDSDSDSKYG DDDSKKASAS APAVDQVVPV ADVQPEPQLG EKLETLESSI TKGPELPGDA 481STAKQSIPFA ITPSSPETVD EKLESSGVSQ DGITTPGQRV VQGKDDIQSS DSDSDSKYGD 541DDSKKASASA PAVDQVVPSD TRADGVSEPL ASHVDQGSDV PGDASVDGVD LRLGRLSTEQ 601SGLLPRHEQN VRAFILEQSL LDQLYMDYID LHPDQKSCEA YNSALHGYNT RLELQKEYNR 661IFESHESASP NEINSFSQKY RAALRDVAQD IVNQGPMFYS SRDAMLLRAR VDTLCDMCRS 721IRNLYMVELD AIDKEEKSLQ SDMKSASSSD KKLIQEKIKL LSEQ ID NO: 9: p16 Antigen Nucleotide Sequence ORIGIN 1ATGTTACACG TTCAAAATCA TGTTGATCAA CATACAAATC ATATAGAACA TGATGATTAC 61CATTTTACTG GTCCTACTAG TTTTGAAGTT AATCTTTCTG AAGAAGAAAA AATGGAGTTA 121CAAGAAGTAT CTTCTATTGA TAGTGTAGGA TGCGAAGATT GTGATCCAAA TTGTCGTTAT 181CCTTTAGAAT TAGTAGAATG TCAGCGTATT GAGGAAAGAC CAGTATGCAA TGCAGGTTTA 241GAGAGCTTGA CTGTTGATGC ATATCAATTA GGATTGTTGT TAGGTGGTTT TTTAAGTGCT 301ATGAATTACA TATCTTATAG CTATCCTTGT TATTATTATG ATTGTTGTGA TAGAAATTAT 361TACGACTGTT GTCATAAGAA TGCGTGTTAT TACAACTGTT GTGATTGTGC GTAASEQ ID NO: 10 p16 Antigen Protein Sequence ORIGIN 1MLHVQNHVDQ HTNHIEHDDY HFTGPTSFEV NLSEEEKMEL QEVSSIDSVG CEDCDPNCRY 61PLELVECQRI EERPVCNAGL ESLTVDAYQL GLLLGGFLSA MNYISYSYPC YYYDCCDRNY 121YDCCHKNACY YNCCDCA.SEQ ID NO: 11 Ribosomal Protein L1 Nucleotide Sequence ORIGIN 1ATGACGATTT TCTTAGAAAG TGATGATGAT AAGAGTAACT TTAAGAAGAC ATTGGAGAAC 61GGTACTAAAG ACAAGACAAA TCTAGATAAT ACTTATTATG ACTATCATCA TGAAGATGAT 121ATGGGAAATA CTGAATATCA TTATGTGAGT TTGGATAGAG TGGATCATGT TAAGATGCCT 181GAAGAGCCTG TAGGTTATGG TGGAGATACT TTACCTATTG TTCCTACTAC AGCTGCTAGT 241GTATCTGGTA GTGATGCAGG CGTTGCTGTA GGTAATGTTA AAGATTTTGA AGATAATGTT 301TTTCATCATA CATCTACTAT AAGAAACGAT GAATTGAAGA TAGATTTACG AATACATACT 361TTAAAGGATT TATCTGATAA AAGATTACGT GAAATTGAAA AGGGATTTAA TGATACGGTA 421ACAAAATTTA AAAATAATTT TGGGTTAGAA CCAAATGATG GAGAAACTAT TTTTGATTTA 481TACCTTTTTG ATGATAAGGA ACAATATAAT TATTATGGAA AGCTTTATAA CTTAGGAATT 541AGTGGATCTG GAGGTATGAC TTTCTATGGA AATGCTAATG TTCCATATAA AATTTATGTA 601CATCAATATG GTGAAATATT GAATTTAAAA CATGAATTAA CTCATGCATT AGAAAGTTAT 661GCATCTGGAC ATAAATTGCA TGGTTCTGAC GTAAATAGCA GAATATTTAC GGAAGGATTA 721GCTGATTATA TCCAAGAAGA TAATAGTTTT ATTATGAGAG GATTAAAGGA TCGAGAGATC 781ACTTCAGATG TATTGAAAGA TTCTTCTGGT AATGTAGATC ATTTAAGTGG TGTTGCAGTG 841AATGAAAATC AGAGGTTAAG TTATAGTATA GGACATGCAT TTGTAAGCTT TTTACAAGAG 901AAATATCCTA AGTTAATTTC GGAATATTTA AACGCATTAA AAGAGGATAA TATTATTCGT 961GCTAAAGAAA TAATTAGTAT GGATAAGTAT CCAGATTTTG AGCCGTGGGT GAAGTCTAAA 1021GACATTAGTT TATATTTAGA AAATATGAAT GTATTAAAGT TAGGATTAGG TGAGAAAATG 1081TTTTCTGCTG AAAGTGCTAG CTATTTTGAA GATCAAGGTG TCAATAAAGA ATATTACCAT 1141GAAAATATTT ATGATATGAG TGGTAAACTA GTAGGTGAAA TGTCACCTGT AGTGCATTAT 1201GCACAAAAAA ATGTGATTCG TATTTGGAAT ATTGCAAGTC CTGATATGAT AGAGGTGCGA 1261CCAGAATATA ACTTTCTGAA ATTGGTAACT ACTCCATCTG GTAAGTCTGC ATATGTATAT 1321TGTGATAAGA ATGGGCATGA GTATTTTAAT ACTAAAGATT ACATAGATTC TGCGTTTAAT 1381ATATTGGCAA GATATGATGT TAAGCTTCGT GAAAGTAGTG ATGCTTTGGA TATTAGAGGT 1441CGTTACTCAG ATGCTGCTAA AGTGTTTAGT AAGCTGCCTA ATGCGGATTT GCTGTTGGAT 1501AAGTTTTTAG AAAAAATAGG TTATAGTAGT TATAAGCAGA TAATAATGAG TAATCCAGAA 1561CAGCTTAATT CTATTAAGGC TTATGTAGTA AAAGAAGTGT TTGAAAATTT TAGGGAATCT 1621GAGGTCAAAA AGGTGTTGAG TGGTGAGTCT CATCCGGAAG TAAGAAATGT ATTAATGGAT 1681CTTACCTATG TTGATTTAAA GAGTGTTATA GGAGTAAATG GTGCAGATAT TGACAGTATT 1741ATTTCTAATC CAGATGTAAT GTTGCGTACT GCTGTGTTAG GTAAAGGAAA TGCAAGTGGG 1801ATATCTCTAT ATGTAGATGA TCAGAAAGTT GGTGAGCTGT CAACTGAAGC AGGTTATTGT 1861GTTAAAAATC TTGATACTGG TAAAGTGTAT TTTATGTTCC ATAATGTTGT TGGAATGATA 1921GCAAGTGGTT ATGAAGACAG AGCATATATG GTTGTATTAG AAAAAGATGG TAAGTTTACT 1981ACTGCTCTAG TTAATAATAT ACAAAAAGCA GCAGATGGAA ATGTTGTATG GGATAATCAA 2041TTTAATCATC CGAATATTAA TAACTTGCAC TCAAATTATA AGGAGCTGTT GTTAAATGAT 2101GCTTCAGTTA AAGATTACTC TCATCTTGCG GATGTGAAAT TTAATAAAGA TGATACAGTA 2161ATTGTTAAAG GTGAATTATT AGATGATAAA GGTACTGTAA GTGTAGATGA TGATGTACAT 2221CGTGCAGTTG TTAAGCATGA TGATCAAATA CTACATCAGT TTAAGAGTAT GTCTTTTTAC 2281ATTACTGAAC CATCAGCTGA TTCAGGTGAC AATTATGGAA GTGATTTTTT CATTTCTGAT 2341GAAGGAAAAA ATCTTAGATT TCAACTTCCT AAAGCTATTA CGCATTTGAA ATTGGTTAAT 2401GTTAATGGAA ATAATAAGTT GGTACCATGT ACTAAAGATG GGAATGAACA TCCTGAAGGT 2461ATGCCATCTG ATTTAACGGA TGAATATAGA TATATAGATC CTATTTTTGC TCATACATTT 2521GAGAAACAAA GTTATTCTAA AAATAGTATT AGTGTTGGGT TAGTGGACTT CAGTAAATAT 2581AAAGAAGGAT CTATGTTTAA ATTACAGCAT TATTCTGATG ATTATCATAT TCATAAGGAT 2641GAACAAGGTA ATGTTATTAG GCCTAATAAC AGATCTTACG TTACAAAAGT GGATTTAGTA 2701TATGATGATA AAGTTATTGG GATGTTGTCT GATAGTATAA ATCAATTTCA GGGTGATATT 2761TTCATTTCTG CAAGCCTTAA TTATAGCCAC AATGATTTTC TTTCATCTAA GTACTTTCAG 2821AAAGTTAATA TTGAGGCGTT AGAAAATGGA ATATATAGTG GAAGATATGA TGTAGGAGAT 2881GGTGACCAAA TAGCAGGTCT TAATACTGAT ACAGGTTATA GTGATAAAGC TATTTTTTAC 2941TTTAAAAATG ATAGCGCATC TACTGATATG CCGGCTAGTG ATGTTACTAC TATTTTACCT 3001TATATAAATG AGCTTTAA SEQ ID NO: 12 Ribosomal Protein L1 Protein SequenceORIGIN 1MTIFLESDDD KSNFKKTLEN GTKDKTNLDN TYYDYHHEDD MGNTEYHYVS LDRVDHVKMP 61EEPVGYGGDT LPIVPTTAAS VSGSDAGVAV GNVKDFEDNV FHHTSTIRND ELKIDLRIHT 121LKDLSDKRLR EIEKGFNDTV TKFKNNFGLE PNDGETIFDL YLFDDKEQYN YYGKLYNLGI 181SGSGGMTFYG NANVPYKIYV HQYGEILNLK HELTHALESY ASGHKLHGSD VNSRIFTEGL 241ADYIQEDNSF IMRGLKDREI TSDVLKDSSG NVDHLSGVAV NENQRLSYSI GHAFVSFLQE 301KYPKLISEYL NALKEDNIIR AKEIISMDKY PDFEPWVKSK DISLYLENMN VLKLGLGEKM 361FSAESASYFE DQGVNKEYYH ENIYDMSGKL VGEMSPVVHY AQKNVIRIWN IASPDMIEVR 421PEYNFLKLVT TPSGKSAYVY CDKNGHEYFN TKDYIDSAFN ILARYDVKLR ESSDALDIRG 481RYSDAAKVFS KLPNADLLLD KFLEKIGYSS YKQIIMSNPE QLNSIKAYVV KEVFENFRES 541EVKKVLSGES HPEVRNVLMD LTYVDLKSVI GVNGADIDSI ISNPDVMLRT AVLGKGNASG 601ISLYVDDQKV GELSTEAGYC VKNLDTGKVY FMFHNVVGMI ASGYEDRAYM VVLEKDGKFT 661TALVNNIQKA ADGNVVWDNQ FNHPNINNLH SNYKELLLND ASVKDYSHLA DVKFNKDDTV 721IVKGELLDDK GTVSVDDDVH RAVVKHDDQI LHQFKSMSFY ITEPSADSGD NYGSDFFISD 781EGKNLRFQLP KAITHLKLVN VNGNNKLVPC TKDGNEHPEG MPSDLTDEYR YIDPIFAHTF 841EKQSYSKNSI SVGLVDFSKY KEGSMFKLQH YSDDYHIHKD EQGNVIRPNN RSYVTKVDLV 901YDDKVIGMLS DSINQFQGDI FISASLNYSH NDFLSSKYFQ KVNIEALENG IYSGRYDVGD 961GDQIAGLNTD TGYSDKAIFY FKNDSASTDM PASDVTTILP YINEL.SEQ ID NO: 13 Type IV Secretory Protein VirD4 Nucleotide Sequence ORIGIN1 ATGGATAGTA TAAGTGCAAA TCACATACGC AATATTTTAT TCCTTGTTTT AGGCGCATTT 61TTTGGACTGG AATTTTGCTT TTATTTATCA GGTGTATTAT TCATCTTAAT GGTCTGGGGA 121CCAAATTACC TAGATTTTAA TGCTATAAAT CCCAGTTTGA GTGATTTTCC AGACAGAATT 181TGGCCAACTA TTTTTGACTA TGTACAACAT TGGTGGAAGA ACCCTTCTGC ATACGATGCA 241GTTTTATTAC TTAAGCTAAT AACGTCATTA TGTACACCAG TAGGTATTCT AAGCATAGTA 301TTATGGAACC TTAGAAATAT ATTATTCGAT TGGAGGCCAT TTAAGAAGAA AGAATCACTG 361CATGGAGATT CAAGATGGGC AACAGAAAAA GATATTCGCA AAATAGGATT ACGTAGTAGA 421AAAGGAATAT TATTAGGGAA AGACAAGAGA GGATATCTCA TTGCAGATGG ATATCAACAT 481GCATTGTTAT TTGCACCAAC TGGATCCGGA AAAGGTGTAG GTTTTGTAAT ACCAAACTTA 541TTATTCTGGG AAGATTCTGT AGTAGTACAC GATATAAAAT TAGAGAACTA TGATCTTACA 601AGTGGGTGGA GAAAAAAAAG GGGACAAGAA GTTTTCGTGT GGAACCCAGC ACAACCTGAC 661GGTATAAGTC ACTGTTACAA CCCATTAGAT TGGATAAGCT CTAAGCCTGG ACAAATGGTA 721GATGATGTAC AAAAAATTGC CAATCTAATA ATGCCTGAAC AAGATTTTTG GTATAACGAA 781GCACGTAGTT TATTTGTAGG AGTAGTATTA TACTTACTAG CAGTACCAGA AAAAGTAAAA 841TCCTTTGGAG AAGTTGTAAG AACAATGCGC AGCGATGACG TAGTCTACAA CTTAGCAGTA 901GTACTAGACA CAATAGGGAA AAAGATTCAC CCAGTTGCAT ACATGAATAT AGCTGCATTT 961TTACAAAAAG CAGACAAAGA ACGCTCAGGT GTTGTATCAA CTATGAACTC ATCTTTAGAA 1021TTATGGGCAA ACCCATTAAT AGATACAGCA ACAGCATCAA GTGATTTTAA TATTCAAGAA 1081TTTAAAAGGA AAAAAGTAAC AGTATATGTT GGATTAACAC CAGATAATTT AACTCGTCTT 1141AGACCTTTAA TGCAGGTATT TTATCAACAA GCTACAGAAT TTTTATGTAG AACTTTACCA 1201TCAGATGATG AACCATATGG TGTACTGTTC TTAATGGATG AGTTTCCAAC ATTAGGAAAA 1261ATGGAGCAAT TTCAAACAGG TATCGCATAT TTCCGTGGAT ATAGAGTTAG ACTATTTTTG 1321ATTATTCAAG ATACTGAACA GCTTAAGGGT ATATATGAAG AAGCAGGAAT GAACTCATTC 1381TTATCAAACT CTACTTATAG AATAACTTTT GCTGCAAATA ATATAGAAAC TGCAAATTTA 1441ATATCACAGT TAATAGGAAA TAAAACTGTT AACCAAGAGT CTTTAAACAG ACCTAAATTT 1501TTAGATTTGA ACCCTGCATC ACGTTCATTA CATATATCAG AAACACAAAG AGCTTTACTA 1561TTACCTCAAG AAGTAATAAT GTTACCCAGA GATGAGCAAA TACTTTTAAT AGAATCTACT 1621TATCCTATAA AATCAAAGAA AATAAAATAC TATGAAGACA AAAATTTTAC AAAAAAACTA 1681TTAAAGAGTA CCTTTGTTCC AACTCAAGAG CCTTATGATC CCAACAAAAC AAAAACAGCA 1741ACAAAAGAAA ACGAAGAACC TATGCCAAGT ATTGAAAGCG ATCTTCCTAA AAATACATCT 1801GACAATACTG AAAACAATAT GGAAGATGGT GCAATGTACA GCAGCATAGA AGAAGATTAT 1861GACGATGATG ATGATGATTT TAATTTTGAA GACTTAGATG AATATATGGA TGAAGAAGAA 1921GATTATGATG ATGAAGAATA TGATGATATA GATTATGATG ATAATAACAA TAGTAATGAG 1981GAGTATGAAG AAGATAATCC AGAAGAAGAT GACAATAGCA ATAATCTAGA CGATGAGGAA 2041GAGGAAGAAG ATAATATTAT AGATTATGAA GATGAAGAAG AATATGATGA TAACATAGAC 2101TACAAAGATG ATGACAATAA CTACAACAAA GATACCACTG ACGATCAAGA CTCAAAAAAA 2161CATAATGAAT AGSEQ ID NO: 14 Type IV Secretory Protein VirD4 Protein Sequence ORIGIN 1MDSISANHIR NILFLVLGAF FGLEFCFYLS GVLFILMVWG PNYLDFNAIN PSLSDFPDRI 61WPTIFDYVQH WWKNPSAYDA VLLLKLITSL CTPVGILSIV LWNLRNILFD WRPFKKKESL 121HGDSRWATEK DIRKIGLRSR KGILLGKDKR GYLIADGYQH ALLFAPTGSG KGVGFVIPNL 181LFWEDSVVVH DIKLENYDLT SGWRKKRGQE VFVWNPAQPD GISHCYNPLD WISSKPGQMV 241DDVQKIANLI MPEQDFWYNE ARSLFVGVVL YLLAVPEKVK SFGEVVRTMR SDDVVYNLAV 301VLDTIGKKIH PVAYMNIAAF LQKADKERSG VVSTMNSSLE LWANPLIDTA TASSDFNIQE 361FKRKKVTVYV GLTPDNLTRL RPLMQVFYQQ ATEFLCRTLP SDDEPYGVLF LMDEFPTLGK 421MEQFQTGIAY FRGYRVRLFL IIQDTEQLKG IYEEAGMNSF LSNSTYRITF AANNIETANL 481ISQLIGNKTV NQESLNRPKF LDLNPASRSL HISETQRALL LPQEVIMLPR DEQILLIEST 541YPIKSKKIKY YEDKNFTKKL LKSTFVPTQE PYDPNKTKTA TKENEEPMPS IESDLPKNTS 601DNTENNMEDG AMYSSIEEDY DDDDDDFNFE DLDEYMDEEE DYDDEEYDDI DYDDNNNSNE 661EYEEDNPEED DNSNNLDDEE EEEDNIIDYE DEEEYDDNID YKDDDNNYNK DTTDDQDSKK 721HNE. SEQ ID NO: 15 MDIDNNNVTTSSTQDKSGNLMEVIMRILNFGNNSDEKVSNEDTKVLVESLQPAVNDNVGNPSSEVGKEEN APEVKAEDLQPAVDGSVEHSSSEVGKKVSETSKEESTPEVKAEDLQPAVDGSIEHSSSEVGEKVSKTSKE ESTPEVKAEDLQPAVDDSVEHSSSEVGEKVSETSKEENTPEVKAEDLQPAVDGSIEHSSSEVGEKVSKTS KEESTPEVKAEDLQPAVDDSVEHSSSEVGEKVSETSKEENTPEVKAEDLQPAVDGSVEHSSSEVGEKVSK TSKEESTPEVKAEDLQPAVDDSVEHSSSEVGEKVSETSKEENTPEVRAEDLQPAVDGSVEHSSSEVGEKV SETSKEESTPEVKAEDLQPAVDSSIEHSSSEVGKKVSETSKEESTPEVKAEDLQPAVDGSVEHSSSEVGE KVSETSKEENTPEVKAEDLQPAVDGSVEHSSSEVGEKVSETSKEENTPEVKAEDLQPAVDGSVEHSSSEV GEKVSETSKEESTPEVKAEDLQPAVDDSVEHSSSEVGEKVSETSKEESTPEVKAEDLQPAVDGSVEHSSS EVGEKVSETSKEESTPEVKAEVQPVADGNPVPLNPMPSIDNIDTNIIFHYHKDCKKGSAVGTDEMCCPVS ELMAGEHVHMYGIYVYRVQSVKDLSGVFNIDHSTCDCNLDVYFVGYNSFTNKETVDLI SEQ ID NO: 16KEENAPEVKAEDLQPAVDGSVEHSSSEVGKKVSETSKEESTPEVKAEDLQPAVDGSIEHSSSEVGEKVSKTSKEESTPEVKAEDLQPAVDDSVEHSSSEVGEKVSETSKEENTPEVKAEDLQPAVDGSIEHSSSEVGEKVSKTSKEESTPEVKAEDLQPAVDDSVEHSSSEVGEKVSETSKEENTPEVKAEDLQPAVDGSVEHSSSEVGEKVSKTSKEESTPEVKAEDLQPAVDDSVEHSSSEVGEKVSETSKEENTPEVRAEDLQPAVDGSVEHSSSEVGEKVSETSKEESTPEVKAEDLQPAVDSSIEHSSSEVGKKVSETSKEESTPEVKAEDLQPAVDGSVEHSSSEVGEKVSETSKEENTPEVKAEDLQPAVDGSVEHSSSEVGEKVSETSKEENTPEVKAEDLQPAVDGSVEHSSSEVGEKVSETSKEESTPEVKAEDLQPAVDDSVEHSSSEVGEKVSETSKEESTPEVKAEDLQPAVDGSVEHSSSEVGEKVSETS KEESTPEVKAESEQ ID NO: 18 E. canis P140-1 (72, 89) CPEVKAEDLQPAVDGSVEHSEQ ID NO: 19 E. canis P140-3 (64, 89) CEVGKEENAPEVKAEDLQPAVDGSVEHSEQ ID NO: 20 E. canis CKEESTPEVKAEDLQPAVDGSVEHSSSEVGKKVSETSSEQ ID NO: 21 XPEVKAEDLQPAVDGSVEHX, wherein X =0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.SEQ ID NO: 22 CMLHVQNHVDQHTNHIEHDDYHFTGPT SEQ ID NO: 23CTNHIEHDDYHFTGPTSFEVNLSEEEKMEL SEQ ID NO: 24 CTGPTSFEVNLSEEEKMELQEVSSIDSSEQ ID NO: 25 XMLXVQNHVDQHTNHIEHDDYHFTXPTWherein the X at position 1 is C or is absent, the X at position 4 is Hor Q and the X at position 25 is D or G. SEQ ID NO: 26XTNHIEHDDYHFTXPTSFEVNLSEEEKMELWherein the X at position 1 is C or is absent and the X at position 14is G or D. SEQ ID NO: 27 XTXPTSFEVNLSEEEKMELQEVSSIDSWherein the X at position 1 is C or is absent, and the X at position 3is G or D. SEQ ID NO: 28 XTNHIEHDDYHFTXPTWherein the X at position 1 is C or is absent, and the X at position 14is G or D. SEQ ID NO: 29 XTXPTSFEVNLSEEEKMELWherein the X at position 1 is C or is absent, and the X at position 3is G or D. SEQ ID NO: 30 XTNHIEHDDYHFTXPTSFEVNLSEXEKMELWherein the X at position 1 is C or is absent, the X at position 14 isG or D, and the X at position 25 is E or G. SEQ ID NO: 31XTXPTSFEVNLSEXEKMELQEVSSIDSWherein the X at position 1 is C or is absent, the X at position 3 is Gor D, the X at position 14 is E or G. SEQ ID NO: 32 XTXPTSFEVNLSEXEKMELWherein the X at position 1 is C or is absent, the X at position 3 is Gor D, and the X at position 14 is G or E. SEQ ID NO: 33XMLXVQNHVDOHTNHIEHDDYHFTXPTSFEVNLSEXEKMELQEVSSIDSWherein the X at position 1 is absent or c, the X at position 4 is H orQ, the X at position 25 is D or G, and the X at position 36 is E or G.

Other embodiments of the invention provide the following polypeptides:

-   -   (a) SEQ ID NO:33, wherein the X at position 1 is absent or C,        the X at position 4 is H or Q, the X at position 25 is D or G,        and the X at position 36 is E or G;    -   (b) Amino acids 1-27 of SEQ ID NO:33, wherein the X at position        1 is C, the X at position 4 is H, the X at position 25 is D or        G;    -   (c) Amino acids 13-41 of SEQ ID NO:33, wherein the X at position        25 is D or G, the X at position 36 is E or G; and a C is        optionally present at the amino terminus;    -   (d) Amino acids 24-49 of SEQ ID NO:33, wherein the X at position        25 is D or G, the X at position 36 is E or G, and a C is        optionally present at the amino terminus;    -   (e) Amino acids 1-27 of SEQ ID NO:33, wherein the X at position        1 is C or absent, and wherein the X at position 25 is D or G;    -   (f) Amino acids 13-41 of SEQ ID NO:33, wherein the X at position        25 is D or G, the X at position 36 is E or G, and a C is        optionally present at the amino terminus;    -   (g) Amino acids 24-49 of SEQ ID NO:33, wherein the X at position        25 is D or G, the X at position 36 is E or G, and a C is        optionally present at the amino terminus;    -   (h) Amino acids 13-27 of SEQ ID NO:33, wherein the X at position        25 is D or G, and a C is optionally present at the amino        terminus;    -   (i) Amino acids 24-41 of SEQ ID NO:33, wherein the X at position        25 is D or G, the X at position 36 is E or G, and a C is        optionally present at the amino terminus;    -   (j) Amino acids 13-41 of SEQ ID NO:33, wherein the X at position        25 is D or G, the X at position 36 is E or G, and a C is        optionally present at the amino terminus;    -   (k) Amino acids 24-49 of SEQ ID NO:33, wherein the X at position        25 is D or G, the X at position 36 is E or G, and a C is        optionally present at the amino terminus;    -   (l) Amino acids 24-41 of SEQ ID NO:33, wherein the X at position        25 is D or G, the X at position 36 is E or G, and a C is        optionally present at the amino terminus.

We claim
 1. A composition comprising: (A) one or more purifiedpolypeptides consisting of SEQ ID NO:22-33; (B) one or more purifiedpolypeptides having at least 95% identity to 15 to 27 contiguous aminoacids of SEQ ID NO:22; 15 to 30 contiguous amino acids of SEQ ID NO:23;15 to 27 contiguous amino acids of SEQ ID NO:24; 15 to 27 contiguousamino acids of SEQ ID NO:25; 15 to 30 contiguous amino acids of SEQ IDNO:26; 15 to 27 contiguous amino acids of SEQ ID NO:27; 15 to 16contiguous amino acids of SEQ ID NO:28; 15 to 19 contiguous amino acidsof SEQ ID NO:29; 15 to 30 contiguous amino acids of SEQ ID NO:30; 15 to27 contiguous amino acids of SEQ ID NO:31; 15 to 19 contiguous aminoacids of SEQ ID NO:32; 15 to 49 contiguous amino acids of SEQ ID NO:33;(C) one or more purified polypeptides comprising SEQ ID NO:22-33,wherein the one or more purified polypeptides are 15 to 75 amino acidsin length; (D) a purified polypeptide set forth in SEQ ID NO:33, whereinthe X at position 1 is absent or C, the X at position 4 is 14 or Q, theX at position 25 is D or G, and the X at position 36 is E or G; (E) apurified polypeptide comprising amino acids 13-41 of SEQ ID NO:33,wherein the X at position 25 is D or G, the X at position 36 is E or G;and a C is optionally present at the amino terminus (SEQ ID NO: 30); (F)a purified polypeptide comprising amino acids 24-49 of SEQ ID NO:33,wherein the X at position 25 is D or G, the X at position 36 is E or G,and a C is optionally present at the amino terminus (SEQ ID NO:31); (G)a purified polypeptide comprising amino acids 1-27 of SEQ ID NO: 33,wherein the X at position 1 is C or absent, and wherein the X atposition 25 is D or G (SEQ ID NO 25); (H) a purified polypeptidecomprising amino acids 13-27 of SEQ ID NO:33, wherein the X at position25 is D or G, and a C is optionally present at the amino terminus (SEQID NO:28); (I) a purified polypeptide comprising amino acids 24-41 ofSEQ ID NO:33, wherein the X at position 25 is D or G, the X at position36 is E or G, and a C is optionally present at the amino terminus (SEQID NO:32); or (J) combinations of (A)-(l), wherein the purifiedpolypeptides of (A)-(J) are: (i) in a multimeric form; (ii) are linkedto a heterologous protein, an indicator reagent, an amino acid spacer,an amino acid linker, a signal sequence, a stop transfer sequence, atransmembrane domain, a protein purification ligand, or a combinationthereof; (iii) are linked to a moiety that enhances an immune response;(iv) are linked to a moiety that facilitates polypeptide stability; or(v) are present in a fusion protein.
 2. A composition comprising one ormore purified polypeptides set forth in: SEQ ID NO:22; SEQ ID NO:23; SEQID NO:24; SEQ ID NO:25, wherein the X at position 1 is C; SEQ ID NO:26,wherein the X at position 1 is C; SEQ ID NO:27, wherein the X atposition 1 is C; SEQ ID NO:28, wherein the X at position 1 is C; SEQ IDNO:29, wherein the X at position 1 is C; SEQ ID NO:30, wherein the X atposition 1 is C; SEQ ID NO:31, wherein the X at position 1 is C; SEQ IDNO:32, wherein the X at position 1 is C; SEQ ID NO:33, wherein the X atposition 1 is C.
 3. The composition of claim 2, wherein the purifiedpolypeptides are in a multimeric form.
 4. The composition of claim 2,wherein the purified polypeptides are linked to a heterologous protein,an indicator reagent, an amino acid spacer, an amino acid linker, asignal sequence, a stop transfer sequence, a transmembrane domain, aprotein purification ligand, or a combination thereof.
 5. Thecomposition of claim 2, wherein the purified polypeptides areimmobilized to a solid support.
 6. The composition of claim 1, whereinthe purified polypeptides are linked to a moiety that enhances an immuneresponse.
 7. The composition of claim 1, wherein the purifiedpolypeptides are linked to a moiety that facilitates polypeptidestability.
 8. The composition of claim 1, wherein the purifiedpolypeptides are present in a fusion protein.
 9. A device comprising theone or more purified polypeptides of claim
 2. 10. A device comprising(A) one or more purified polypeptides consisting of SEQ ID NO 22-33; (B)one or more purified polypeptides having at least 95% identity to 15 to27 contiguous amino acids of SEQ ID NO:22; 15 to 30 contiguous aminoacids of SEQ ID NO:23; 15 to 27 contiguous amino acids of SEQ ID NO:24;15 to 27 contiguous amino acids of SEQ ID NO:25; 15 to 30 contiguousamino acids of SEQ ID NO:26; 15 to 27 contiguous amino acids of SEQ IDNO:27; 15 to 16 contiguous amino acids of SEQ ID NO:28; 15 to 19contiguous amino acids of SEQ ID NO:29; 15 to 30 contiguous amino acidsof SEQ ID NO:30; 15 to 27 contiguous amino acids of SEQ ID NO:31 ; 15 to19 contiguous amino acids of SEQ ID NO:32; 15 to 49 contiguous aminoacids of SEQ ID NO:33; (C) one or more purified polypeptides comprisingSEQ ID NO 22-33, wherein the one or more purified polypeptides are 15 to75 amino acids in length; (D) a purified polypeptide set forth in SEQ IDNO:33, wherein the X at position 1 is absent or C, the X at position 4is 14 or Q, the X at position 25 is D or G, and the X at position 36 isE or G; (E) a purified polypeptide comprising amino acids 13-41 of SEQID NO:33, wherein the X at position 25 is D or G, the X at position 36is E or G; and a C is optionally present at the amino terminus (SEQ IDNO:30); (F) a purified polypeptide comprising amino acids 24-49 of SEQID NO: 33, wherein the X at position 25 is D or G, the X at position 36is E or G, and a C is optionally present at the amino terminus (SEQ IDNO: 31); (G) a purified polypeptide comprising amino acids 1-27 of SEQID NO:33, wherein the X at position 1 is C or absent, and wherein the Xat position 25 is D or G (SEQ ID NO:25); (H) a purified polypeptidecomprising amino acids 13-27 of SEQ ID NO: 33, wherein the X at position25 is D or G, and a C is optionally present at the amino terminus (SEQID NO 28); (I) a purified polypeptide comprising amino acids 24-41 ofSEQ ID NO:33, wherein the X at position 25 is D or G, the X at position36 is E or G, and a C is optionally present at the amino terminus (SEQID NO:32); or (J) combinations of (A)-(l), wherein the purifiedpolypeptides of (A)-(J) are bound to a solid support.
 11. A kitcomprising: one or more of: (A) one or more purified polypeptidesconsisting of SEQ ID NO 22-33; (B) one or more purified polypeptideshaving at least 95% identity to 15 to 27 contiguous amino acids of SECID NO:22; 15 to 30 contiguous amino acids of SEC ID NO:23; 15 to 27contiguous amino acids of SEC ID NO:24; 15 to 27 contiguous amino acidsof SEC ID NO:25; 15 to 30 contiguous amino acids of SEC ID NO:26; 15 to27 contiguous amino acids of SEC ID NO:27; 15 to 16 contiguous aminoacids of SEC ID NO:28; 15 to 19 contiguous amino acids of SEC ID NO:29;15 to 30 contiguous amino acids of SEC ID NO:30; 15 to 27 contiguousamino acids of SEC ID NO:31; 15 to 19 contiguous amino acids of SEC IDNO:32; 15 to 49 contiguous amino acids of SEC ID NO:33; (C) one or morepurified polypeptides comprising SEQ ID NO:22-33, wherein the one ormore purified polypeptides are 15 to 75 amino acids in length; (D) apurified polypeptide set forth in SEQ ID NO:33, wherein the X atposition 1 is absent or C, the X at position 4 is H or Q, the X atposition 25 is D or G, and the X at position 36 is E or G; (E) apurified polypeptide comprising amino acids 13-41 of SEQ ID NO:33,wherein the X at position 25 is D or G, the X at position 36 is E or G;and a C is optionally present at the amino terminus (SEQ ID NO:30); (F)a purified polypeptide comprising amino acids 24-49 of SEQ ID NO:33,wherein the X at position 25 is D or G, the X at position 36 is E or G,and C is optionally present at the amino terminus (SEQ ID NO:31); (G) apurified polypeptide comprising amino acids 1-27 of SEQ ID NO:33,wherein the X at position 1 is C or absent, and wherein the X atposition 25 is D or G (SEQ ID NO:25); (H) a purified polypeptidecomprising amino acids 13-27 of SEQ ID NO:33, wherein the X at position25 is D or G, and a C is optionally present at the amino terminus (SEQID NO:28); (I) a purified polypeptide comprising amino acids 24-41 ofSEQ ID NO:33, wherein the X at position 25 is D or G, the X at position36 is E or G, and a C is optionally present at the amino terminus (SEQID NO:32); or (J) combinations of (A)-(l), and (i) a solid support; (ii)one or more antibodies or antibody fragments that specifically bind tothe one or more polypeptides of the composition of A-J; (iii) buffers;(iv) stabilizers; (v) positive controls; (vi) negative controls; (vii)detector reagents; or (viii) combinations of (i)-(viii).